Bioactive peptides and methods of using same

ABSTRACT

Disclosed are peptide ligands for G-protein coupled receptors that are useful for treating disorders associated with G-protein coupled receptor activation.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/923,690 filed on Jun. 21, 2013 which is a divisional of U.S.application Ser. No. 12/668,712 filed on Jun. 7, 2010 which is aNational Stage of International Application No. PCT/IB2008/002447 filedon Jul. 11, 2008, which in turn claims priority to U.S. ProvisionalApplication No. 60/959,370, filed Jul. 12, 2007. The contents of theseapplications are hereby incorporated by reference in their entireties.

FIELD OF THE INVENTION

The invention relates to bioactive peptides.

BACKGROUND OF THE INVENTION

Known and uncharacterized GPCRs currently constitute major targets fordrug action and development. There are ongoing efforts to identify new Gprotein coupled receptors and to deorphanize known GPCRs, which can beused to screen for new agonists and antagonists having potentialprophylactic and therapeutical properties.

SUMMARY OF THE INVENTION

The invention is based in part on the identification of novel peptides,variants, homologs, orthologs or derivatives thereof:

Peptide P59-S-Amide (Amide): (SEQ ID NO: 1) AYAAFSV-Amide; PeptideP59-SG (free acid Gly) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide)(SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid)(SEQ ID NO: 4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide)(SEQ ID NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide): (SEQID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74 (free acid)(SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V (amide)(SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG;that are ligands for the relaxin-related GPCRs, selected from the groupconsisting of RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 and RXFP4; and/or of theLGR family GPCRs, selected from a group consisting of but not limited toLRR (A10) containing GPCRs: FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3),LGR4, LGR5, LGR6, LGR7 (RXFP1) and LGR8 (RXFP2).

In one aspect, the invention features peptides less than 100 amino acidsin length, wherein the peptide comprises the amino acid sequence ofFormula I:

X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶-X¹⁷-X¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³-X²⁴-X²⁵-X²⁶-X²⁷-X²⁸-X²⁹-X³⁰-X³¹-X³²-X³³

Wherein

X¹ is absent or G or a small naturally or non-naturally occurring aminoacid;

X² is absent or Q or a polar naturally or non-naturally occurring aminoacid;

X³ is absent or K or a basic naturally or non-naturally occurring aminoacid;

X⁴ is absent or G or a small naturally or non-naturally occurring aminoacid;

X⁵ is absent or Q or S a polar naturally or non-naturally occurringamino acid;

X⁶ is absent or V or A or P or M or a hydrophobic naturally ornon-naturally occurring amino acid;

X⁷ is absent or G or a small naturally or non-naturally occurring aminoacid;

X⁸ is absent or P or L or A naturally or non-naturally occurring aminoacid;

X⁹ is absent or P or Q naturally or non-naturally occurring amino acid;

X¹⁰ is absent or G or a small naturally or non-naturally occurring aminoacid;

X¹¹ is absent or A or H or E or D or a hydrophobic or a small or anacidic naturally or non-naturally occurring amino acid;

X¹² is absent or A or P or Q or S or R or H or a hydrophobic or a smallnaturally or non-naturally occurring amino acid;

X¹³ is absent or C or V or a hydrophobic naturally or non-naturallyoccurring amino acid;

X¹⁴ is absent or R or K or Q or P or a basic or a polar naturally ornon-naturally occurring amino acid;

X¹⁵ is absent or R or Q or S or a basic or a polar naturally ornon-naturally occurring amino acid;

X¹⁶ is absent or A or L or H or Q or a hydrophobic or a small naturallyor non-naturally occurring amino acid;

X¹⁷ is absent or Y or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid;

X¹⁸ is absent or A or a hydrophobic or small naturally or non-naturallyoccurring amino acid;

X¹⁹ is absent or A or a hydrophobic small naturally or non-naturallyoccurring amino acid;

X²⁰ is absent or F or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid;

X²¹ is absent or S or T or a polar naturally or non-naturally occurringamino acid;

X²² is absent or V or a hydrophobic naturally or non-naturally occurringamino acid;

X²³ is absent or G or hydrophobic or small non-naturally occurring aminoacid or replaced by an Amide;

X²⁴ is absent or R or a basic naturally or non-naturally occurring aminoacid;

X²⁵ is absent or R or a basic naturally or non-naturally occurring aminoacid;

X²⁶ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X²⁷ is Y or a hydrophobic or an aromatic naturally or non-naturallyoccurring amino acid;

X²⁸ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X²⁹ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X³⁰ is F or a hydrophobic naturally or non-naturally occurring aminoacid;

X³¹ is S or T or a polar naturally or non-naturally occurring aminoacid;

X³² is V or a hydrophobic naturally or non-naturally occurring aminoacid;

X³³ is absent or G or hydrophobic or small naturally or non-naturallyoccurring amino acid or replaced by an Amide;

or a pharmaceutically acceptable salt thereof.

In some embodiments, the amino acid sequence of said peptides differesby at least one, or at least two, or at least three amino acids ascompared to the naturally occurring amino acid sequence set forth in SEQID NOs:1-15, 20-26.

In some embodiments, the peptide of this invention has at leastone-forth, e.g., one third, one half, or the same activity, as theactivity of a peptide of substantially identical length with a naturallyoccurring amino acid sequence. By “substantially identical length” ismeant the same length or a difference in length of nomore than tenpercent.

In some embodiments, the peptide binds to a G-protein coupled receptor(GPCR) protein.

In some embodiments, the GPCR protein belongs to a relaxin-relatedfamily of GPCR proteins selected from the group consisting ofrelaxin-related GPCRs, including RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 andRXFP4; and/or of the LGR family of GPCRs, selected from a groupconsisting of but not limited to LRR (A10) containing GPCRs: FSHR(LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5, LGR6, LGR7 and LGR8.

In some embodiments, the peptide inhibits forskolin-mediated increasesin cAMP levels in an LG7R7 and/or LGR8 expressing CHO-K1 cell.

In some embodiments, the peptide is a degradation product of a naturallyoccurring protein isolated from a cell. In other embodiments, thepeptide is isolated from a protein recombinantly produced in a cell. Thecell can be, e.g., a prokaryotic or eukaryotic cell.

In some embodiments, the peptide is chemically synthesized in vitro.

If desired, the he peptide can be provided coupled to a biotin moiety.

In some embodiments, the peptide includes a disulfide bond.

The peptide can be provided as a linear or cyclic peptide. In someembodiments, the peptide is a cyclic lactam. In some embodiments, thepeptide is a branched peptide.

In some embodiments, the peptide is phosphorylated, e.g., at a serine,theronine, or tyrosine residue.

In some embodiments, a cysteine residue in a Peptide P59 or Peptide P73sequence is replaced with a second amino acid, e.g., one that preventsdimerization. Examples of suitable amino acids include leucine,isoleucine, alanine, and valine.

In some embodiments, the peptide is modified at its amino terminus.Example of amino terminal modifications include an N-glycated,N-alkylated, N-acetylated or N-acylated amino acid.

In some embodiments, the peptide is pegylated.

In some embodiments, the peptide includes a C-terminal amidated aminoacid. In other embodiments, the peptide does not include an amidatedamino acid at its carboxy terminus.

In some embodiments, the non-naturally occurring amino acid is anomega-amino acid, e.g. beta-alanine (beta-Ala), or 3 aminopropionic(3-aP).

In some embodiment, the peptide includes a small non-naturally occurringamino acid, e.g., sarcosine (Sar), β-alanine (β-Ala), 2,3diaminopropionic (2,3-diaP) or alpha-aminisobutyric acid (Aib);omega-acid beta-alanine (beta-Ala), or 3 aminopropionic (3-aP) acid

In some embodiments, the peptide includes a hydrophobic non-naturallyoccurring amino acid, e.g., t butylalanine (t BuA), t butylglycine (tBuG), N methylisoleucine (N MeIle), norleucine (Nle), methylvaline(Mvl), cyclohexylalanine (Cha), phenylglycine (Phg), NaI,β2-thienylalanine (Thi), 2 naphthylalanine (2 Nal), or1,2,3,4-tetrahydroisoquinoline-3 carboxylic acid (Tic).

In some embodiments, the peptide includes a basic non-naturallyoccurring amino acid, e.g., ornithine (Orn) or homoarginine (Har).

In some embodiments, the peptide includes a neutral and polarnon-naturally occurring amino acid, e.g., citrulline (Cit), Acetyl Lys,or methionine sulfoxide (MSO).

In some embodiments, the peptide is less than 225, 200, 175, 150, 125,100, 75, 50, 30, 25, 20, 15, 10, 9, 8, 7, 6, or 5 amino acids.

Peptides can be modified to include one or more modifications. Thus, insome embodiments, the carboxy terminus is amidated and an internalcysteine is replaced with a valine at position 13 (C13V) in Formula I,above. In other embodiments, both modifications are present:

Peptide P-59S (amide) (SEQ ID NO: 1) AYAAFSV-Amide; Peptide P-59S (freeacid) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide) (SEQ ID NO: 3)GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO: 4)GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID NO: 5)GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid) (SEQ ID NO: 6)GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (amide) (SEQ ID NO: 7)GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Amide Peptide P74 (free acid) (SEQ IDNO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V-Amide (amide)(SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV. PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said peptide is conjugated or fused to a secondpeptide or polypeptide, optionally wherein said second peptide orpolypeptide are multiple antigenic peptides (MAP), or wherein saidsecond peptide or polypeptide comprises a portion of an immunoglobulin,or wherein said second peptide or polypeptide comprises albumin or aportion of albumin.

The invention in another embodiment includes any one of the foregoingpeptides, wherein said second peptide or polypeptide includes a signalsequence.

The invention in another embodiment includes any one of the foregoingpeptides, wherein signal sequence comprises: MAAPALLLLALLLPVGA (SEQ IDNO:11),

(SEQ ID NO: 12) MAAPALLLLALLLPVGAWP, (SEQ ID NO: 13)MAAPALLLLALLLPVGAWPGLP.

The invention in another embodiment includes a pharmaceuticalcomposition comprising any one of the foregoing peptides and apharmaceutically acceptable carrier.

The invention in another embodiment includes a peptide comprising afragment of any one of the foregoing peptides, wherein said peptidefragment binds or activates a G-protein coupled receptor (GPCR) protein,optionally wherein said GPCR protein belongs to the family of proteins,selected from the group consisting of relaxin-related GPCRs, includingRXFP1 (LGR7), RXFP2 (LGR8), RXFP3 and RXFP4; and/or of the LGR family ofGPCRs, selected from a group consisting of but not limited to LRR (A10)containing GPCRs: FSHR (LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5,LGR6, LGR7 and LGR8.

The invention in another embodiment includes a purified nucleic acidsequence encoding any one of the foregoing peptides, variants,homoplogus, orthologus or derivatives thereof.

The invention in another embodiment includes a method of treating adisorder where modulation or activation of a relaxin-related GPCR and/orLGR family of GPCR receptor or receptors is efficacious and/or oftherapeutic value, the method comprising administering to the subject atherapeutically effective amount of a peptide of Formula I. The disorderis selected from but not limited to hyperplastic disorders, neoplasticdisorders, cancer; fibrotic conditions, disorders of collagendeposition, fibrotic breakdown, connective tissue remodeling,uncontrolled or abnormal collagen or fibronectin formation or breakdown;skin injuries including wound healing and scarring, scleroderma;urogenital disorders including female reproductive disorders, male andfemale infertility, cryptorchidism, disregulation of spermatogenesis andreproductive development including descent of the gonads; conditionsassociated with pregnancy such as preeclampsia or complication of labor;angiogenesis related disorders; cardiovascular disorders,vasodilatation, vasoconstriction or hypertension, endothelialdisfunction and vascular disease, congestive heart failure, coronaryartery disease, ischemia and ischemia-reperfusion, peripheral vasculardisease; kidney disease, renal disease associated with arteriosclerosisor other narrowing of kidney capillaries; capillaries narrowing in thebody, such as in the eyes or in the peripheral digits, the mesocaecum,lung and peripheral vasculature; CNS related disorders, neurologicaldisorders, cognition and memory related indications, depression,neurological modification; inflammatory disorders, such as gastritis,gout, gouty arthritis, arthritis, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, ulcers; autoimmunedisorders; inflammatory conditions associated with viral infection andinfection related diseases including fibrosis and cirrhosis; Raynaud'sdisease, Raynaud's phenomenon; bone related conditions includingosteoporosis; metabolic disorders including food and water intake,diabetes, obesity; respiratory or a pulmonary disorder, includingasthma, COPD, bronchial disease, lung diseases, cystic fibrosis, ARDS,SARS.

The invention in another embodiment includes a method of treating ahypertension and its complications, said method comprising administeringto a subject in need thereof a therapeutically effective amount of anyone of the foregoing peptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said hypertension associated disorder is selected from the groupconsisting of hypertension and its complications including but notlimited to hypertensive heart disease; antihypertension (blood pressurereduction); systemic and pulmonary high blood pressure; cerebrovasculardisease and stroke; heart failure and stroke; left ventricularhypertrophy (LVH); congestive heart failure (CHF); hypertension, highblood pressure; vasodilation; renal hypertension; diuresis; nephritis;natriuresis; scleroderma renal crisis; angina pectoris (stable andunstable); myocardial infarction; heart attack; coronary artery disease;coronary heart disease; cardiac arrhythmias; atrial fibrillation; portalhypertension; raised intraocular pressure; vascular restenosis; chronichypertension; valvular disease; myocardial ischemia; acute pulmonaryedema; acute coronary syndrome; hypertensive retinopathy; hypertensivepregnancy sickness; preeclampsia; Raynaud's phenomenon; erectiledysfunction, glaucoma. These peptides are also used as a vasodilator andin antithrombotic therapy

The invention in another embodiment includes a method of treating acardiovascular diseases and their-complications, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of any one of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said cardiovascular disorder is selected from a group consistingof peripheral vascular diseases and coronary artery diseases, includingbut not limited to myocardial infarction; congestive heart failure(CHF); myocardial failure; myocardial hypertrophy; ischemiccardiomyopathy; systolic heart failure; diastolic heart failure; stroke;thrombotic stroke; concentric LV hypertrophy, myocarditis;cardiomyopathy; hypertrophic cardiomyopathy; myocarditis; decompensatedheart failure; ischemic myocardial disease; congenital heart disease;angina pectoris; prevention of heart remodeling or ventricularremodeling after myocardial infarction; ischemia-reperfusion injury inischemic and post-ischemic events (e.g. myocardial infarct);cerebrovascular accident; mitral valve regurgitation; hypertension;hypotension; restenosis; fibrosis; thrombosis; or platelet aggregation.

The invention in another embodiment includes a method of treating afibrotic condition in a subject, involving tissue remodeling followinginflammation or ischemia-reperfusion injury, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said fibrotic conditions is selected from a group consisting offibrotic conditions involving tissue remodeling following inflammationor ischemia-reperfusion injury, including but not limited toendomyocardial and cardiac fibrosis fibrosis; mediastinal fibrosis;idiopathy pulmonary fibrosis; pulmonary fibrosis; retroperitonealfibrosis; fibrosis of the spleen; fibrosis of the pancreas; hepaticfibrosis (cirrhosis) alcohol and non-alcohol related (including viralinfection such as HAV, HBV and HCV); fibromatosis; granulomatous lungdisease; glomerulonephritis myocardial scarring following infarction;endometrial fibrosis and endometriosis; wound healing whether by injuryor surgical procedures, diabetes related wound fibrosis.

The invention in another embodiment includes a method of treating aendothelial dysfunction disease in a subject, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said endothelial dysfunction disease is selected from a groupconsisting of cardiovascular diseases, high blood pressure,atherosclerosis, thrombosis, myocardial infarct, heart failure, renaldiseases, plurimetabolic syndrome, erectile dysfunction; vasculitis; anddiseases of the central nervous system (CNS).

The invention in another embodiment includes a method of treating arespiratory disease in a subject, said method comprising administeringto a subject in need thereof a therapeutically effective amount ofanyone of the foregoing peptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said respiratory disease is selected from a group consisting ofincluding but not limited to asthma, bronchial disease, lung diseases,chronic obstructive pulmonary disease (COPD), Acute Respiratory DistressSyndrome (ARDS), severe acute respiratory syndrome (SARS), Fibrosisrelated Asthma, cystic fibrosis.

The invention in another embodiment includes a method of preventing ortreating a skin injury or tissue repair, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said skin injury is selected from a group including but notlimited to dermal repair, wound healing; burns, erythemas, lesions,wound healing following surgical procedures; skin or tissue lesionsincluding lesions induced by conditions including, but not limited toPsoriasis, Lupus and Kaposhi Sarcoma; Scleroderma and collagenousdiseases of the skin and skin tumors.

The invention in another embodiment includes a method of treating aurogenital disorder or a genitor-urological disorder in a subject, saidmethod comprising administering to a subject in need thereof atherapeutically effective amount of anyone of the foregoing peptides,wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said urogenital disorder or genitor-urological disorders isselected from group consisting of urogenital disorder or agenitor-urological disorders including but not limited to renal disease;a bladder disorder; disorders of the reproductive system; gynecologicdisorders; urinary tract disorder; incontinence; disorders of the male(spermatogenesis, spermatic motility), and female reproductive system;sexual dysfunction; erectile dysfunction; embryogenesis; and conditionsassociated with pregnancy. These are also used in pregnancy monitoring.As used herein, the term “conditions associated with pregnancy”includes, but is not limited to, conditions of fertilisation, pregnancy,parturition and lactation. The invention in another embodiment includesusing the peptides of the invention falling within Formulas I, such asfor example, peptides as depicted in SEQ ID NOs:6-7, 9-16, 18-25, 27-31,wherein said pregnancy related disorders are selected from a groupconsisting of Abnormal endometrial angiogenesis; Placental developmentdefects; Cervical ripening (softening); Abnormal implantation; Nippledevelopment and disfunction; Pregnancy related remodeling of the Uterinetissue; Endometriosis; Preeclampsia; Lactation disorders; Estrogenic andnon-estrogenic related hormonal disorders; Pre-term labor; post termlabor; and Labor complications.

The invention in another embodiment includes a method of treating acancer or inflammation associated with cancer in a patient, said methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of anyone of the foregoing peptides, wherein saidpeptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said cancer is selected from a group consisting of solid cancer,including but not limited to colon cancer, lung cancer, breast cancer,prostate cancer, brain cancer, pancreatic cancer, ovarian cancer, kidneycancer, testicular cancer, bone cancer, osteosarcoma, or liver cancer(HBV/HCV related or non-related). The cancer can alternatively be amelanoma, glioma, a sarcoma, a leukemia, or lymphoma. These peptides arealso useful in the prevention or treatment of invasive and metastaticcancer.

The invention in another embodiment includes a method of treating a boneand bone related disorders in a patient, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said bone disease is selected from a group including but notlimited to Osteoporosis; Osteoarthritis; Osteopetrosis; Boneinconsistency; Osteosarcoma; and Cancer matastesis to the bone.

The invention in another embodiment includes a method of treating ametabolic and metabolic related disorders in a patient, said methodcomprising administering to a subject in need thereof a therapeuticallyeffective amount of anyone of the foregoing peptides, wherein saidpeptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said metabolic disorder is selected from a group including butnot limited to diabetes, diabetes mellitus, lipodystrophy,hyperthyroidism, glaucoma, hyperlipidaemia, non-insulin dependentdiabetes, Food intake; Water intake; Feeding and drinking behaviors,Anorexia, Cachexia (cancer and non cancer related); Fat and lipidmetabolism; and Energy control, appetite control and obesity.

The invention in another embodiment includes a method of treating a CNSand cognition disorders in a patient, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said CNS and cognition disorders are selected from a groupincluding but not limited to central and peripheral degenerativeneuropathies; neuroprotection; impaired cognition; anxiety disorders,pain control, food intake, a behavioral disorder, a learning disorder, asleep disorder, a memory disorder, a pathologic response to anesthesia,addiction, depression, migraine, a menstruation disorder, muscle spasm,opiate dependence, dementia, Alzheimer's disease, Parkinson's disease,cortical function, locomotor activity, Alcohol and Drug addiction andabuse; Impared memory; Feeding and drinking related behaviours; Stresscontrol, Bipolar disorder; Schyzophrenia; Schyzoaffective; MultipleSclerosis (MS); Stroke and stroke damage repair (Ischemia protection);Vasculature and re-vasculature in the brain; and Brain tissueregeneration and a peripheral nervous system disorder.

The invention in another embodiment includes a method of treatingischemia-reperfusion injury associated with ischemic and post-ischemicevents in organs and tissues in a patient, said method comprisingadministering to a subject in need thereof a therapeutically effectiveamount of anyone of the foregoing peptides, wherein said peptide isFormula I.

The invention in another embodiment includes the foregoing method,wherein said ischemia-reperfusion injury associated with ischemic andpost-ischemic events in organs and tissues disorders are selected from agroup including but not limited to thrombotic stroke; myocardialinfarction; angina pectoris; embolic vascular occlusions; peripheralvascular insufficiency; splanchnic artery occlusion; arterial occlusionby thrombi or embolisms, arterial occlusion by non-occlusive processessuch as following low mesenteric flow or sepsis; mesenteric arterialocclusion; mesenteric vein occlusion; ischemia-reperfusion injury to themesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation.

The invention in another embodiment includes a method of treatingischemia-reperfusion injury following conditions including but notlimited to procedures such as cardiac surgery; organ surgery; organtransplantation; angiography; cardiopulmonary and cerebral resuscitationin a patient, said method comprising administering to a subject in needthereof a therapeutically effective amount of anyone of the foregoingpeptides, wherein said peptide is Formula I.

The invention in another embodiment includes a method of inflammatoryconditions associated with an infection, e.g., a bacterial infection ora viral infection in a patient, said method comprising administering toa subject in need thereof a therapeutically effective amount of anyoneof the foregoing peptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said inflammatory conditions associated with an infection, areselected from but not limited to including but not limited to a viralinfection caused by human immunodeficiency virus I (HIV-1) or HIV-2,acquired immune deficiency (AIDS), West Nile encephalitis virus,coronavirus, rhinovirus, influenza virus, dengue virus, HCV, HBV, HAV,hemorrhagic fever; an otological infection; severe acute respiratorysyndrome (SARS), sepsis and sinusitis.

The invention in another embodiment includes a method of kidney diseasesin a patient, said method comprising administering to a subject in needthereof a therapeutically effective amount of anyone of the foregoingpeptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said kidney diseases are selected from but not limited todiabetic nephropathy; glomerulosclerosis; nephropathies; renalimpairment; scleroderma renal crisis and chronic renal failure.

The invention in another embodiment includes a method of angiogenesisrelated conditions in a patient, said method comprising administering toa subject in need thereof a therapeutically effective amount of anyoneof the foregoing peptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said angiogenesis related conditions are selected from but notlimited to retinal angiogenesis in a number of human ocular diseasessuch as diabetes mellitus, retinopathy of prematury, and age-relatedmacular degeneration, or cancer associated angiogenesis in primary ormetastatic cancer, including but not limited to cancer of the prostate,brain, breast, colorectal, lung, ovarian, pancreatic, renal, cervical,melanoma, soft tissue sarcomas, lymphomas, head-and-neck, andglioblastomas.

The invention in another embodiment includes a method of inflammatorydisorder in a patient, said method comprising administering to a subjectin need thereof a therapeutically effective amount of anyone of theforegoing peptides, wherein said peptide is Formula I.

The invention in another embodiment includes the foregoing method,wherein said inflammatory disorder are selected from but not limited togastritis, gout, gouty arthritis, arthritis, rheumatoid arthritis,inflammatory bowel disease, Crohn's disease, ulcerative colitis, ulcers,chronic bronchitis, asthma, allergy, acute lung injury, pulmonaryinflammation, airway hyper-responsiveness, vasculitis, septic shock andinflammatory skin disorders, including but not limited to psoriasis,atopic dermatitis, eczema.

The invention in another embodiment includes a method of autoimmunedisorder in a patient, said method comprising administering to a subjectin need thereof a therapeutically effective amount of anyone of theforegoing peptides, wherein said peptide is Formula I. The invention inanother embodiment includes the foregoing method, wherein saidautoimmune disorder are selected from but not limited to multiplesclerosis, psoriasis, rheumatoid arthritis, systemic lupuserythematosus, ulcerative colitis, Crohn's disease, transplantrejection, immune disorders associated with graft transplantationrejection, benign lymphocytic angiitis, lupus erythematosus, Hashimoto'sthyroiditis, primary myxedema, Graves' disease, pernicious anemia,autoimmune atrophic gastritis, Addison's disease, insulin dependentdiabetes mellitis, Good pasture's syndrome, myasthenia gravis,pemphigus, sympathetic ophthalmia, autoimmune uveitis, autoimmunehemolytic anemia, idiopathic thrombocytopenia, primary biliarycirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren'ssyndrome, rheumatic disease, polymyositis, scleroderma, mixed connectivetissue disease, inflammatory rheumatism, degenerative rheumatism,extra-articular rheumatism, collagen diseases, chronic polyarthritis,psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoidarthritis, periarthritis humeroscapularis, panarteriitis nodosa,progressive systemic scleroderma, arthritis uratica, dermatomyositis,muscular rheumatism, myositis, myogelosis and chondrocalcinosis.

The invention in another embodiment includes the cDNA that encodes thepeptide sequences of the invention, which can be used in gene therapy.

If desired, gene therapy can be used to deliver to a subject a peptideaccording to the invention. A nucleic acid encoding the peptide can beinserted into vectors, which are then used as gene therapy vectors. Genetherapy vectors can be delivered to a subject by, for example,intravenous injection, local administration or by stereotacticinjection. The pharmaceutical preparation of the gene therapy vector caninclude the gene therapy vector in an acceptable diluent, or cancomprise a slow release matrix in which the gene delivery vehicle isimbedded. Alternatively, where the complete gene delivery vector can beproduced intact from recombinant cells, e.g., retroviral vectors, thepharmaceutical preparation can include one or more cells that producethe gene delivery system.

The invention in another embodiment includes combination therapy usingone or more peptides of the present invention provided in combinationwith another therapeutic agent or agents. As used herein, the term“combination therapy” refers to treatment of a single condition ordisease involving the concomitant use of more than one therapeuticagent.

The invention in another embodiment includes an antibody thatselectively binds to an epitope in anyone of the foregoing peptides.

In a still further aspect, the invention provides an antibody thatselectively binds to an epitope in the peptide of Formula I.

In some embodiments, the antibody binds selectively and/or is raised tothe following sequences:

Peptide P-59S-Amide (amide) (SEQ ID NO: 1) AYAAFSV-Amide; Peptide P-59S(free acid) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide) (SEQ IDNO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid) (SEQ ID NO:4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide) (SEQ ID NO: 5)GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid) (SEQ ID NO: 6)GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (amide) (SEQ ID NO: 7)GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Amide Peptide P74 (free acid) (SEQ IDNO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V-Amide (amide)(SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV. PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG.

The invention in another embodiment includes anyone of the foregoingantibodies, wherein the antibody is a monoclonal antibody.

The invention in another embodiment includes anyone of the foregoingantibodies, wherein the antibody is conjugated or coupled to adetectable label, a radioactive label, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, or a therapeutic agent.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the invention, suitable methods and materials aredescribed below. All publications, patent applications, patents, andother references mentioned herein are incorporated by reference in theirentirety. In the case of conflict, the present Specification, includingdefinitions, will control. In addition, the materials, methods, andexamples are illustrative only and not intended to be limiting.

Other features and advantages of the invention will be apparent from thefollowing detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the Gi (cAMP inhibition) effect of P59C13V(P59) and P74C13V (P74) on forskolin-treated LGR7 and LGR8 transientlytransfected CHO-K1 cells.

FIG. 2 presents a Gi (cAMP inhibition) dose response curve atconcentrations between 1 nM to 10 uM of P59C13V (P59), P74C13V (P74) andINSL3 on CHO-K1 cells transiently transfected with LGR8 (RXFP2) andpre-treated with 20 uM of Forskolin.

FIG. 3A is a graph showing a Gs (cAMP increase) assay following notreatment or treatment with HCG (control), P59C13V (P59) and P74C13V(P74), respectively on LGR4, LGR5, and LGR6 transfected CHO-K1 cells.

FIG. 3B is a graph showing a Gi (cAMP inhibition) assay following pretreatment by 20 uM of Forskolin followed by no treatment or treatmentwith HCG (control), P59C13V (P59), and P74C13V (P74), respectively onLGR5, transfected CHO-K1 cells.

FIG. 3C is a graph showing a Gi (cAMP inhibition) assay following pretreatment by 20 uM of Forskolin followed by no treatment or treatmentwith HCG (control), P59C13V (P59), and P74C13V (P74), respectively onLGR4, transfected CHO-K1 cells.

FIG. 4A is a graph showing cAMP dose response activation followingtreatment pre-treated with 20 uM of Forskolin and treatment withincreasing doses (1 nM-10 uM) of P59C13V (P59), P74C13V (P74) orrelaxin, on CHO-K1 cells transiently transfected with LGR7.

FIG. 4B is a graph showing Gs (cAMP increase) dose response activationfollowing treatment with low concentrations (1-100 nM) of P59C13V (P59)or relaxin on CHO-K1 cells transiently transfected with LGR7.

FIG. 5 is a graph showing Gs (cAMP increase) dose respose activation ofEuroscreen recombinant cell lines expressing the RXFP3 receptor (Cat:D88437, assay cat: ES-656-MG). The cAMP dose response was comparedbetween Relaxin 3 (positive control) and P59C13V (P59) at concentrationsof 1 pM-1 uM for Relaxin 3 and 1 nM-10 uM for P59C13V (P59).

FIGS. 6A-B are graphs showing cAMP responsive element reading (CRE) inresponse to stimulation of LGR8 expressing HEK293 cells to INSL3 (as apositive control) and the peptides P59-S amide and P74C13V (P74) (FIG.6A); or P59C13V (P59) and P59C13V-amide (P59-amide)) (FIG. 6B). The Xaxis represents the peptide concentration (log) and the Y axisrepresents the CRE activation relative to INSL3 max activity (in %).

FIGS. 7A-B are graphs showing cAMP responsive element reading (CRE) inresponse to stimulation of LGR7 expressing CHO-K1 cells to pre treatmentwith 5 uM of Forskolin and H2 relaxin (H2) as a positive controlcompared with the peptides P59-S amide and P74C13V (P74) (FIG. 7A); orP59C13V (P59) and P59C13V-amide (P59-amide) (FIG. 7B). The X axisrepresents the peptide concentration (log) and the Y axis represents theCRE activation relative to the base line Forskolin activity (in % where100% is with no peptide treatment). This graph is the average result ofthree independent experiments (N=3).

FIGS. 8A-B are graphs showing cAMP responsive element reading (CRE) inresponse to stimulation of LGR8 expressing CHO-K1 cells to pre treatmentwith 5 uM of Forskolin and INSL3 as a positive control compared with thepeptides P59-S amide and P74C13V (P74) (FIG. 8A); or P59C13V (P59) andP59C13V-amide (P59-amide) (FIG. 8B). The X axis represents the peptideconcentration (log) and the Y axis represents the CRE activationrelative to the base line Forskolin activity (in % where 100% is with nopeptide treatment). This graph is the average result of threeindependent experiments (N=3).

FIG. 9 is a graph showing competitive binding of LGR7 between a Europium(radioactive) conjugated Relaxin (H2) and either non-radioactive H2relaxin and each of the peptides: P59-S-amide, P74C13V (P74), P59C13V(P59) and P59C13V-amide (P59-amide). The X axis represents the peptideconcentration (log) and the Y axis represents the specific bindingrepresented by radioactivity (%).

FIG. 10 is a graph showing competitive binding of LGR8 between aEuropium (radioactive) conjugated INSL3 and either non-radioactive INSL3and each of the peptides: P59-S-amide, P74C13V (P74), P59C13V (P59) andP59C13V-amide (P59-amide). The X axis represents the peptideconcentration (log) and the Y axis represents the specific bindingrepresented by radioactivity (%).

FIG. 11 is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) of H2 Relaxin (1 uM),P74C13V (P74-10 uM), 0.1% BSA (as a negative control) and 1 uM ofCalcitonin (a ligand for CalcR a GPCR endogenously expressed on CHO-K1cells, as a positive internal control) on Untransfected CHO-K1 cells.The X axis represents time of experiment (challenge time is indicated bya vertical line), and the Y axis represents the cell index (ameasurement of the change in Cell impedance) normalized to the peptidechallenge time. The code is as follows: circles represent calcitonin 1uM; short horizontal lines represents H2 1 uM; squares represent P74 10uM; and diamond shapes represent BSA 0.1%.

FIG. 12 is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) of H2 Relaxin (1 uM),P74C13V (P74-10 uM), 0.1% BSA (as a negative control) and 1 uM ofCalcitonin (a ligand for CalcR a GPCR endogenously expressed on CHO-K1cells, as a positive internal control) on GPR39 (a non relaxin relatedGPCR) transfected CHO-K1 cells. The X axis represents time of experiment(challenge time is indicated by a vertical line), and the Y axisrepresents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time. The code is asfollows: circles represent calcitonin 1 uM; diamond shapes representP74, 10 uM; squares represent H2, 1 uM; and short horizontal linesrepresent BSA 0.1%.

FIG. 13 is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) of H2 Relaxin (1 uM),P74C13V (P74-10 uM), 0.1% BSA (as a negative control) and 1 uM ofCalcitonin (a ligand for CalcR a GPCR endogenously expressed on CHO-K1cells, as a positive internal control) on LGR7 (RXFP1) transfectedCHO-K1 cells. The X axis represents time of experiment (challenge timeis indicated by a vertical line), and the Y axis represents the cellindex (a measurement of the change in Cell impedance) normalized to thepeptide challenge time. The code is as follows: diamond shapes representP74, 10 uM; circles represent H2, 1 uM; short horizontal lines representcalcitonin 1 uM; and squares represent BSA 0.1%.

FIG. 14 is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) of H2 Relaxin (1 uM),P74C13V (P74-10 uM), P59C13V (P59), 0.1% BSA (as a negative control) and1 uM of Calcitonin (a ligand for CalcR a GPCR endogenously expressed onCHO-K1 cells, as a positive internal control) on LGR8 (RXFP2)transfected CHO-K1 cells. The X axis represents time of experiment(challenge time is indicated by a vertical line), and the Y axisrepresents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time. The code is asfollows: diamond shapes represent P59, 1 uM; circles represent H2, 1 uM;short horizontal lines represent calcitonin 1 uM; squares represent P74,10 uM; and triangles represent BSA 0.1%.

FIG. 15 is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) of H2 Relaxin (1 uM),P74C13V (P74-10 uM), 0.1% BSA (as a negative control) and 1 uM ofCalcitonin (a ligand for CalcR a GPCR endogenously expressed on CHO-K1cells, as a positive internal control) on LGR4 (Orphan) transfectedCHO-K1 cells. The X axis represents time of experiment (challenge timeis indicated by a vertical line), and the Y axis represents the cellindex (a measurement of the change in Cell impedance) normalized to thepeptide challenge time. The code is as follows: circles represent P74,10 uM; diamond shapes represent H2, 1 uM; squares represent calcitonin 1uM; and short horizontal lines represent BSA 0.1%.

FIGS. 16A-B is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) as a dose dependantresponse of different concentrations of H2 Relaxin on LGR7 (RXFP1)transfected CHO-K1 cells. FIG. 16A: The X axis represents time ofexperiment (challenge time is indicated by a vertical line), and the Yaxis represents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time (indicated by theleft vertical line). Each line represents a different concentration(according to legend). FIG. 16B: a dose response curve as calculated bythe ACEA RT-CES system according to the results in FIG. 16A. The X axisrepresents the peptide's concentration in log of M. The Y axisrepresents the normalized cell index at the end point of the experiment(indicated by the right vertical line in FIG. 16A). The code for FIG.16A is as follows: 0.16 nM; 0.8 nM; 4 nM; 20 nM; 100 nM; and 500 nM arerepresented by black circles; triangles; squares; diamond-shapes; whitecircles; and horizontal lines, respectively.

FIGS. 17A-B is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) as a dose dependantresponse of different concentrations of P74C13V (P74) on LGR7 (RXFP1)transfected CHO-K1 cells. FIG. 17A: The X axis represents time ofexperiment (challenge time is indicated by a vertical line), and the Yaxis represents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time (indicated by theleft vertical line). Each line represents a different concentration(according to legend). FIG. 17B: a dose response curve as calculated bythe ACEA RT-CES system according to the results in FIG. 17A. The X axisrepresents the peptide's concentration in log of M. The Y axisrepresents the normalized cell index at the end point of the experiment(indicated by the right vertical line in FIG. 17A). The code for FIG.17A is as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM arerepresented by black circles; triangles; squares; diamond-shapes; whitecircles; and horizontal lines, respectively.

FIG. 18A-B is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) as a dose dependantresponse of different concentrations of P59C13V (P59) on LGR7 (RXFP1)transfected CHO-K1 cells. FIG. 18A: The X axis represents time ofexperiment (challenge time is indicated by a vertical line), and the Yaxis represents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time (indicated by theleft vertical line). Each line represents a different concentration(according to legend). FIG. 18B: a dose response curve as calculated bythe ACEA RT-CES system according to the results in FIG. 18A. The X axisrepresents the peptide's concentration in log of M. The Y axisrepresents the normalized cell index at the end point of the experiment(indicated by the right vertical line in FIG. 18A). The code for FIG.18A is as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM arerepresented by black circles; triangles; squares; diamond-shapes; whitecircles; and horizontal lines, respectively.

FIG. 19A-B is a graph showing the cell impedance and cytoskeletonremodeling effect (measured by ACEA RT-CES system) as a dose dependantresponse of different concentrations of P59S-Amide on LGR7 (RXFP1)transfected CHO-K1 cells. FIG. 18A: The X axis represents time ofexperiment (challenge time is indicated by a vertical line), and the Yaxis represents the cell index (a measurement of the change in Cellimpedance) normalized to the peptide challenge time (indicated by theleft vertical line). Each line represents a different concentration(according to legend). FIG. 19B: a dose response curve as calculated bythe ACEA RT-CES system according to the results in FIG. 19A. The X axisrepresents the peptide's concentration in log of M. The Y axisrepresents the normalized cell index at the end point of the experiment(indicated by the right vertical line in FIG. 19A). The code for FIG.19A is as follows: 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; and 10 uM arerepresented by black circles; triangles; squares; diamond-shapes; whitecircles; and horizontal lines, respectively.

FIG. 20: This figure shows a multiple alignment of the sequence of P59-G(SEQ ID No. 12) as a part of the native precursor (C1QTNF8—SEQ. ID. No.19), including the flanking dibasic (K or R) cleavage sites, withhomologous sequences derived from different organisms, includingChimpanzee (SEQ ID No. 20), Orangutan (SEQ ID No. 21), Rhesus (SEQ IDNo. 22), Cow (SEQ ID No. 23), Chicken (SEQ ID No. 24) and Rat (SEQ IDNo. 26), and the corresponding peptide sequence from the human paralogueC1QTNF1 (SEQ ID No. 25) (all sequences include the flanking dibasiccleavage sites). As can be seen both the N-terminal end (4 Amino acids)and C-terminal end (7 Amino acids) are identical to all species. Thecleavage sites at both ends are also highly conserved (with occasionalreplacement of K for an R). The middle Cysteine residue (C13) that wasreplaced with Valine for dimerization purposes also is highly conserved.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides bioactive peptides. These peptides are useful,inter alia, for treating a variety of indications and disorders, whichare discussed in detail below. In some embodiments, the peptides areligands for GPCR receptors.

The peptides disclosed herein are related to GPCR ligand of the LGR andRelaxin family:

-   -   1. The LGR family: consists of all LRR containing GPCRs        including LGR1 (FSHR), LGR2 (LHCGR), LGR3 (TSHR), KGR4, LGR5,        LGR6, LGR7 (RXFP1) and LGR8 (RXFP2).    -   2. The Relaxin related family: consists of all Relaxin activated        receptors including RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135)        and RXFP4 (GPCR142).

LGR8 (RXFP2) (SwissProt accession number: RXFP2_HUMAN) is a loweraffinity receptor for relaxin (H2 and H1). The activity of this receptoris mostly but not exclusively mediated by G proteins leading tostimulation of adenylate cyclase and an increase of cAMP. Binding of theligand activates a TRK pathway. LGR8 is also a high affinity receptorfor the Leydig insulin-like 3 peptide (INSL3). LGR8 belongs to theG-protein coupled receptor 1 family. It contains 1 LDL-receptor class Adomain and 10 LRR (leucine-rich) repeats. LGR8 is expressed in thebrain, kidney, muscle, thyroid, testis, placenta, uterus, ovary,adrenal, prostate, skin, peripheral blood cells, bone marrow,osteoblasts and heart. Defects in LGR8 are a cause of cryptorchidism,also known as impaired testicular descent, one of the most frequentcongenital abnormalities in humans, involving 2-5% of male births.Cryptorchidism is associated with increased risk of infertility andtesticular cancer. LGR8 is involved in CNS related diseases likeAlzheimer (Shen P J., et al., Ann N Y Acad Sci. 2005; 1041:510-5).Stimulation with relaxin increases cell proliferation, invasiveness, andadhesion in vitro. The suppression of relaxin decreased cellinvasiveness by 90% and growth by 25% and increased cell apoptosis up by2.2 times (Feng S, et al. Clin. Cancer Res. (2007). LGR8 is alsoinvolved in Osteoporosis, Female infertility and non cryptorchidismrelated male infertility.

LGR7 (RXFP1) (SwissProt Accession Number: RXFP1_HUMAN) is a receptor forrelaxin. LGR7 binds all human relaxin peptides with high affinity, buthas very low affinity for INSL3. The activity of this receptor ismediated by G proteins leading both to stimulation of adenylate cyclaseand an increase of cAMP, and inhibition of adenylate cyclase and adecrease of cAMP depending on the tissue and cell type. Binding of theligand activates a TRK pathway that inhibits the activity of aphosphodiesterase that degrades cAMP. LGR7 belongs to the G-proteincoupled receptor 1 family. It contains 1 LDL-receptor class A domain and10 LRR (leucine-rich) repeats. LGR7 is expressed in the brain, liver,kidney, testis, breast placenta, uterus, uterus endometrium, cervix,vagina, ovary, adrenal, prostate, skin, osteoclasts and heart. The LGR7receptor is regulated by Estrogen and the activity of Estrogenreceptors. LGR7 is involved in many processes including Collagendeposition and scaring, Pregnancy and labor processes includingimplantation and cervical ripening, Vasodilatation, Tumor progressionand invasiveness, food intake and Osteoporosis.

LGR4 (SwissProt Accession Number: LGR4_HUMAN; G-protein coupled receptor48 (GPR48)) is an orphan seven trans-membrane receptor that belongs tothe G-protein coupled receptor 1 family. It contains 15 LRR(leucine-rich) repeats. LGR4 is expressed in multiple steroidogenictissues: placenta, ovary, testis and adrenal. It is also expressed inspinal cord, thyroid, stomach, trachea, heart, pancreas, kidney,prostate and spleen.

LGR5 (SwissProt Accession Number: LGR5_HUMAN; G-protein coupled receptor49; G-protein coupled receptor 67 (GPR49, GPR67)) is an orphan seventrans-membrane receptor that belongs to the G-protein coupled receptor 1family. It contains 17 LRR (leucine-rich) repeats. LGR5 is prediocted tobe an important receptor for signals controlling growth anddifferentiation of specific embryonic tissues [Morita H, et al. Mol CellBiol. 2004 November; 24(22):9736-43].

LGR6 (SwissProt Accession Number: LGR6_HUMAN) is an orphan seventrans-membrane receptor that belongs to the G-protein coupled receptor 1family. It contains 16 LRR (leucine-rich) repeats. LGR6 is expressed inmultiple steroidogenic tissues: placenta, ovary, testis, and adrenal. Itis also expressed in spinal cord, thyroid, stomach trachea, heart,pancreas, kidney, prostate and spleen.

GPCR135 (RXFP3) (SwissProt Accession Number: RL3R1_HUMAN; Relaxin-3receptor 1, RLN3 receptor 1, Relaxin family peptide receptor 3,Somatostatin- and angiotensin-like peptide receptor, G protein-coupledreceptor SALPR, GPCR135), is the receptor for relaxin-3 (H3 Relaxin).The RXFP3 receptor also has a lower affinity for Relaxin 2 (H2 Relaxin).Binding of the ligand inhibits cAMP accumulation by coupling with Giproteins. This receptor is Expressed predominantly in brain regions.Highest expression in substantia nigra and pituitary, followed byhippocampus, spinal cord, amygdala, caudate nucleus and corpus callosum,quite low level in cerebellum. In peripheral tissues, relatively highlevels in adrenal glands, low levels in pancreas, salivary gland,placenta, mammary gland and testis. Defects in this receptor's activity,as well as reduction in its primary ligand Relaxin 3 cause a decrease inbody weight and feeding behaviour in mice.

GPCR142 (RXFP4) (SwiisProt Accession Number: RL3R2_HUMAN; Relaxin-3receptor 2, Relaxin family peptide receptor 4, G-protein coupledreceptor 100, GPCR142), is the receptor for INSL5. This receptor is alsoactivated by Relaxin 3, Relaxin 2 as well as bradykinin and kallidin.Binding of the ligand inhibits cAMP accumulation by coupling with Giproteins. This receptor is expressed in a broader range of tissuesincluding brain, kidney, testis, thymus, placenta, prostate, salivarygland, thyroid and colon.

The relaxin ligands superfamily currently comprises 10 members with arelatively high degree of sequence homology. These family membersinclude insulin, insulin-like grown factors I and II, relaxin 1, 2 and3, and the insulin-like hormones INSL3, 4, 5 and 6. The relaxinsuperfamily members have a wide range of biological activities which arewell described in the art.

The actions of relaxin (mostly 1 and 2) include an ability to inhibitmyometrial contractions, stimulation of remodelling of connective tissueand induction of softening of the tissues of the cervix and birth canal.Additionally, relaxin (1, 2) increases growth and differentiation of themammary gland and nipple and induces the breakdown of collagen (mostlyby induction of MMP proteins that breakdown ECM components such ascollagen as well as inhibition of TIMP proteins which induce ECM, suchas collagen, synthesis), one of the main components of connective tissueas well as fibrotic tissues. Relaxin decreases collagen synthesis andincreases the release of collagenases. Female mice lacking afunctionally active relaxin gene failed to relax and elongate theinterpubic ligament of the pubic symphysis and could not suckle theirpups, which in turn, died within 24 hours unless cross-fostered torelaxin wild type or relaxin heterozygous foster mothers.

Relaxin (1 and 2) has additionally been reported to cause a widening ofblood vessels (vasodilatation) in the kidney, mesocaecum, lung andperipheral vasculature, which leads to increased blood flow or perfusionrates in these tissues. It also stimulates an increase in heart rate andcoronary blood flow, and increases both glomerular filtration rate andrenal plasma flow. Relaxin (1,2) has also been found to inhibithistamine release and the accumulation of calcium, as well as promotenitric oxide synthesis, during cardiac anaphylaxis.

The brain is another target tissue for relaxin where the peptides havebeen shown to bind to receptors in the circumventricular organs toaffect blood pressure, food intake and drinking

Relaxin (1 and 2) has also been implicated in depression of plateletaggregation and their release by megakaryocytes, and may thus beassociated with clotting disorders.

The INSL peptides, such as INSL3, has been shown to be involved inmaturation and descent of the testes, as well as the survival of spermcells, development of ovarian follicles and maturation of the oocyte.Therefore, potential clinical applications of INSL3 agonists andantagonists include the treatment of fertility disorders, or the controlof fertility levels.

INSL3 has been implicated in regulation of relaxin activity in theheart. Thus, INL3 agoinsits of the invention can are useful for treatingheart disease. Furthermore, INSL3 polymorphisms have been hyperplasticand neoplastic disorders of the thyroid gland, suggesting a role forthis relaxin superfamily member in the etiology of these pathologies.

Provided by the invention are bioactive peptides falling within FormulaI.

Formula I includes compounds falling within the following formula:

Wherein

X¹ is absent or G or a small naturally or non-naturally occurring aminoacid;

X² is absent or Q or a polar naturally or non-naturally occurring aminoacid;

X³ is absent or K or a basic naturally or non-naturally occurring aminoacid;

X⁴ is absent or G or a small naturally or non-naturally occurring aminoacid;

X⁵ is absent or Q or S a polar naturally or non-naturally occurringamino acid;

X⁶ is absent or V or A or P or M or a hydrophobic naturally ornon-naturally occurring amino acid;

X⁷ is absent or G or a small naturally or non-naturally occurring aminoacid;

X⁸ is absent or P or L or A naturally or non-naturally occurring aminoacid;

X⁹ is absent or P or Q naturally or non-naturally occurring amino acid;

X¹⁰ is absent or G or a small naturally or non-naturally occurring aminoacid;

X¹¹ is absent or A or H or E or D or a hydrophobic or a small or anacidic naturally or non-naturally occurring amino acid;

X¹² is absent or A or P or Q or S or R or H or a hydrophobic or a smallnaturally or non-naturally occurring amino acid;

X¹³ is absent or C or V or a hydrophobic naturally or non-naturallyoccurring amino acid;

X¹⁴ is absent or R or K or Q or P or a basic or a polar naturally ornon-naturally occurring amino acid;

X¹⁵ is absent or R or Q or S or a basic or a polar naturally ornon-naturally occurring amino acid;

X¹⁶ is absent or A or L or H or Q or a hydrophobic or a small naturallyor non-naturally occurring amino acid;

X¹⁷ is absent or Y or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid;

X¹⁸ is absent or A or a hydrophobic or small naturally or non-naturallyoccurring amino acid;

X¹⁹ is absent or A or a hydrophobic small naturally or non-naturallyoccurring amino acid;

X²⁰ is absent or F or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid;

X²¹ is absent or S or T or a polar naturally or non-naturally occurringamino acid;

X²² is absent or V or a hydrophobic naturally or non-naturally occurringamino acid;

X²³ is absent or G or hydrophobic or small naturally or non-naturallyoccurring amino acid or replaced by an Amide;

X²⁴ is absent or R or a basic naturally or non-naturally occurring aminoacid;

X²⁵ is absent or R or a basic naturally or non-naturally occurring aminoacid;

X²⁶ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X²⁷ is Y or a hydrophobic or an aromatic naturally or non-naturallyoccurring amino acid;

X²⁸ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X²⁹ is A or a hydrophobic or small naturally or non-naturally occurringamino acid;

X³⁰ is F or a hydrophobic naturally or non-naturally occurring aminoacid;

X³¹ is S or T or a polar naturally or non-naturally occurring aminoacid;

X³² is V or a hydrophobic naturally or non-naturally occurring aminoacid;

X³³ is absent or G or hydrophobic or small naturally or non-naturallyoccurring amino acid or replaced by an Amide;

or a pharmaceutically acceptable salt thereof.

In some embodiments, a peptide of Formula I includes the amino acidsequence of one of the following:

Peptide P59-S-Amide (Amide): (SEQ ID NO: 1) AYAAFSV-Amide; PeptideP59-SG (free acid Gly) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide)(SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid)(SEQ ID NO: 4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide)(SEQ ID NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide): (SEQID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74 (free acid)(SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V (amide)(SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG,

that are ligands for the relaxin-related GPCRs selected from the groupconsisting of RXFP1 (LGR7), RXFP2 (LGR8), RXFP3 (GPCR135) and RXFP4(GPCR142); and/or of the LGR family of GPCRs, selected from a groupconsisting of but not limited to LRR containing GPCRs: FSHR (LGR1),LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5, LGR6, LGR7 (RXFP1) and LGR8(RXFP2).

The present invention also encompasses polypeptides encoded by thepolynucleotide sequences of the present invention, as well aspolypeptides according to the amino acid sequences described herein.

The present invention also encompasses homologues of these polypeptides,such homologues can be at least 50%, at least 55%, at least 60%, atleast 65%, at least 70%, at least 75%, at least 80%, at least 85%, atleast 85%, at least 90%, at least 95% or more say 100% homologoues tothe amino acid sequence set forth below, as can be determined usingBlastP software of the National Center of Biotechnology Information(NCBI) using default parameters, optionally and preferably including thefollowing: filtering on (thisoption filters repetitive or low-complexitysequences from the query using the Seg (protein) program), scoringmatrix is BLOSUM62 for proteins, word size is 3, E value is 10, gapcosts are 11, 1 (initialization and extension). Optionally andpreferably, nucleic acid sequence identity/homology is determined withBlastN software of the National Center of Biotechnology Information(NCBI) using default parameters, which preferably include using the DUSTfilter program, and also preferably include having an E value of 10,filtering low complexity sequences and a word size of 11. Finally thepresent invention also encompasses fragments of the above describedpolypeptides and polypeptides having mutations, such as deletions,insertions or substitutions of one or more amino acids, either naturallyoccurring or artificially induced, either randomly or in a targetedfashion.

The term “homolog” relating to a peptide of the invention as used hereinshould be understood to encompass a peptide which has substantially thesame amino acid sequence and substantially the same biological activityas peptides depicted in SEQ ID NOs: 1-15, 20-26. Thus, a homolog maydiffer from the peptides depicted in SEQ ID NOs: 1-15, 20-26 by theaddition, deletion or substitution of one or more amino acid residues,provided that the resulting peptide retains the biological activity ofpeptides depicted in SEQ ID NOs: 1-15, 20-26, respectively. Personsskilled in the art can readily determine which amino acid residues maybe added, deleted or substituted (including with which amino acids suchsubstitutions may be made) using established well known procedures.Examples of homologs of peptides depicted in SEQ ID NOs: 1-15, 20-26 aredeletion homologs containing less than all the amino acid residues ofpeptides depicted in SEQ ID NOs: 1-15, 20-26, respectively, substitutionhomologs wherein one or more amino acid residues specified are replacedby other amino acid residues (eg. amino acid with similar properties orby D-amino acids, or by non-natural amino acids) and addition homologswherein one or more amino acid residues are added to a terminal ormedial portion of peptides depicted in SEQ ID NOs: 1-15, 20-26,respectively.

The term “derivative” relating to a peptide of the invention should beunderstood to encompass a peptide which has substantially the same aminoacid sequence and substantially the same biological activity as peptidesdepicted in SEQ ID NOs: 1-15, 20-26, respectively. Thus, a derivativemay differ from the peptides depicted in SEQ ID NOs: 1-15, 20-26 by amodification, such as but not limited to glycosylation, amidation,acetylation, alkylation, alkenylation, alkynylation, phosphorylation,sulphorization, hydroxylation, hydrogenation and so forth. Thus, aderivative of a peptide of the invention may differ from the peptidesdepicted in SEQ ID NOs: 1-15, 20-26 by a modification on one or moreamino acid residues, provided that the resulting peptide retains thebiological activity of peptides depicted in SEQ ID NOs: 1-15, 20-26,respectively. Persons skilled in the art can readily determine whichamino acid residues may be modified using established well knownprocedures. In one embodiment, a peptide of the invention is amidated atits C-terminus and acetylated at its N-terminus.

By “variant” is meant a polypeptide that differs from a referencepolypeptide, but retains essential properties. Generally, differencesare limited so that the sequences of the reference polypeptide and thevariant are closely similar overall and, in many regions, identical. Avariant and reference polypeptide may differ in amino acid sequence byone or more substitutions, additions, and/or deletions, in anycombination. A substituted or inserted amino acid residue may or may notbe one encoded by the genetic code. A variant of a polypeptide may be anaturally occurring such as an allelic variant, or it may be a variantthat is not known to occur naturally. Non-naturally occurring variantsof polypeptides may be made by mutagenesis techniques or by directsynthesis.

Generally, the variant differs from the reference polypeptide byconservative amino acid substitutions, whereby a residue is substitutedby another with like characteristics (e.g. acidic, basic, aromatic,etc.). Typical substitutions are among Ala, Val, Leu and Ile; among Serand Thr; among the acidic residues Asp and Glu; among Asn and Gln; andamong the basic residues Lys and Arg; or aromatic residues Phe and Tyr.

“A peptide with substantially the same biological activity” as usedherein should be understood to encompass a peptide which has at leasthas at least one-forth, e.g., one third, one half, or the same activity,as the activity of a peptide of substantially identical length with anaturally occurring amino acid sequence. By “substantially identicallength” is meant the same length or a difference in length of no morethan ten percent.

A peptide within Formula I can be provided as part of a longer peptidethat includes the specified amino acid sequence. For example, thepeptide can be provided on a peptide that is less than 200, 150, 125,100, 75, 50, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 13, 11,10, 9, 8 or 7 amino acids.

In preferred embodiments, the peptide fragment retains one or more ofthe activities associated with the full-length peptide, e.g., binding toand/or activation of a GPCR receptor, or activity against a conditiondescribed herein. In some embodiments, a peptide within Formula I bindsa G-protein coupled receptor (GPCR) protein, which is preferablyrelaxin-related GPCR or LGR family GPCR. In a further preferredembodiments the relaxin-related GPCR is selected from the groupconsisting of but not limited to RXFP1, RXFP2, RXFP3 or RXFP4. or fromthe LGR related family group consisting of but not limited to FSHR(LGR1), LHCGR (LGR2), TSHR (LGR3), LGR4, LGR5, LGR6 LGR7 (RXFP1) and/orLGR8 (RXFP2) proteins.

In some embodiments, a peptide within Formula I activates a GPCRprotein. Activation of a GPCR protein can be measured using methodsknown in the art.

A peptide within Formula I can be provided conjugated to a secondpeptide or polypeptide. Examples of second peptides or polypeptides aremultiple antigenic peptides (MAP) and a signal sequence. Suitable signalsequences include, e.g.

(SEQ ID NO: 16) MAAPALLLLALLLPVGA, (SEQ ID NO: 17) MAAPALLLLALLLPVGAWP,(SEQ ID NO: 18) MAAPALLLLALLLPVGAWPGLP.

In some embodiments, the second peptide or polypeptide is animmunoglobulin sequence (e.g., an IgG sequence). Immunoreactive ligandsfor use as a targeting moiety in the invention include anantigen-recognizing immunoglobulin (also referred to as “antibody”), orantigen-recognizing fragment thereof, e.g., immunoglobulins that canrecognize a tumor-associated antigen. As used herein, “immunoglobulin”refers to any recognized class or subclass of immunoglobulins such asIgG, IgA, IgM, IgD, or IgE.

Preferred are those immunoglobulins which fall within the IgG class ofimmunoglobulins. The immunoglobulin can be derived from any species.Preferably, however, the immunoglobulin is of human, murine, or rabbitorigin. In addition, the immunoglobulin may be polyclonal or monoclonal,but is preferably monoclonal.

Conjugates of the invention may include an antigen-recognizingimmunoglobulin fragment. Such immunoglobulin fragments may include, forexample, the Fab′, F(ab′) 2, Fv or Fab fragments, or otherantigen-recognizing immunoglobulin fragments. Such immunoglobulinfragments can be prepared, for example, by proteolytic enzyme digestion,for example, by pepsin or papain digestion, reductive alkylation, orrecombinant techniques. The materials and methods for preparing suchimmunoglobulin fragments are well-known to those skilled in the art. SeeParham, J. Immunology, 131, 2895, 1983; Lamoyi et al., J. ImmunologicalMethods, 56, 235, 1983.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to amino acid polymers in which one or more amino acidresidue is an analog or mimetic of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Theterms encompass any peptide (including cyclic peptides) or proteincomprising two or more amino acids joined to each other by peptide bondsor modified peptide bonds. “Polypeptide” refers to both short chains,commonly referred to as peptides, oligopeptides or oligomers, and tolonger chains, generally referred to as proteins.

“Polypeptides” include amino acid sequences modified either by naturalprocesses, or by chemical modification techniques which are well knownin the art. Modifications may occur anywhere in a polypeptide, includingthe peptide backbone, the amino acid side-chains, and the amino orcarboxyl termini. Polypeptides can be modified, e.g., by the addition ofcarbohydrate residues to form glycoproteins. The terms “polypeptide,”“peptide” and “protein” include glycoproteins, as well asnon-glycoproteins.

Peptides within the invention can be produced using methods known in theart, e.g., by purifying the peptide sequence from a naturally occurringprotein or peptide. Purification can be performed along with a cleavageor degradation (either enzymatic or non-enzymatic) to produce thedesired peptide using methods known in the art.

Alternatively, products can be biochemically synthesized using, e.g.,solid phase synthesis, partial solid phase synthesis methods, fragmentcondensation, classical solution synthesis. These methods are preferablyused when the peptide is relatively short (i.e., 10 kDa) and/or when itcannot be produced by recombinant techniques (i.e., not encoded by anucleic acid sequence).

Solid phase polypeptide synthesis procedures are well known in the artand further described by John Morrow Stewart and Janis Dillaha Young,Solid Phase Peptide Syntheses (2nd Ed., Pierce Chemical Company, 1984).

Synthetic polypeptides can be purified by preparative high performanceliquid chromatography [Creighton T. (1983) Proteins, structures andmolecular principles. WH Freeman and Co. N.Y.] and the composition ofwhich can be confirmed via amino acid sequencing.

Polypeptides or peptides can alternatively be synthesized usingrecombinant techniques such as those described by Bitter et al., (1987)Methods in Enzymol. 153:516-544, Studier et al. (1990) Methods inEnzymol. 185:60-89, Brisson et al. (1984) Nature 310:511-514, Takamatsuet al. (1987) EMBO J. 6:307-311, Coruzzi et al. (1984) EMBO J.3:1671-1680 and Brogli et al., (1984) Science 224:838-843, Gurley et al.(1986) Mol. Cell. Biol. 6:559-565 and Weissbach & Weissbach, 1988,Methods for Plant Molecular Biology, Academic Press, NY, Section VIII,pp 421-463.

A peptide within the invention may include one or more modifications.For example, it may be provided phosphorylated (typically at a serine,threonine, or tyrosine residue), pegylated, coupled to a biotin moiety,or include a disulfide bond to another peptide, polypeptide or aminoacid. The peptide may be provided in a cyclic form, e.g., as a cyclicpeptide or as a lactam. Alternatively, or in addition, the peptide maybe provided as a branched peptide.

The peptide may be additionally modified (when linear) at its aminoterminus or carboxy terminus. Examples of amino terminal modificationsinclude, e.g., N-glycated, N-alkylated, N-acetylated or N-acylated aminoacid. A terminal modification can include a pegylation. An example of acarboxy terminal modification is a c-terminal amidated amino acid.

A peptide of the invention may contain amino acids other than the 20gene-encoded amino acids. When amino acids are not designated as eitherD- or L-amino acids, the amino acid is either an L-amino acid or couldbe either a D- or L-amino acid, unless the context requires a particularisomer.

The notations used herein for the polypeptide amino acid residues arethose abbreviations commonly used in the art. The less commonabbreviations Abu, Cpa, Nle, Pal, Tle, Dip, 4-Fpa, and Nal stand for2-amino-butyric acid, p-chloroPhenylalanine, norleucine,3-pyridyl-2-alanine, tert-leucine, 2,2-diphenylalanine,4-fluoro-phenylalanine, and 3-(2-naphthyl)-alanine or3-(1-naphthyl)-alanine, respectively.

One example of a non-naturally occurring amino acid is an omega-aminoacid, e.g., beta-alanine (beta-Ala), or 3 aminopropionic (3-aP). Otherexamples are non-naturally occurring amino acids, e.g., sarcosine (Sar),β-alanine (β-Ala), 2,3 diaminopropionic (2,3-diaP) oralpha-aminisobutyric acid (Aib); omega-acid is beta-alanine (beta-Ala),or 3 aminopropionic (3-aP); a hydrophobic non-naturally occurring aminoacid, such as t-butylalanine (t BuA), t butylglycine (t BuG), Nmethylisoleucine (N MeIle), norleucine (Nle), methylvaline (Mvl),cyclohexylalanine (Cha), phenylglycine (Phg), NaI, β2-thienylalanine(Thi), 2 naphthylalanine (2 Nal), or 1,2,3,4-tetrahydroisoquinoline-3carboxylic acid (Tic); a basic amino acid, such as ornithine (Orn) orhomoarginine (Har); and a neutral/polar non-naturally occurring aminoacid is citrulline (Cit), Acetyl Lys, or methionine sulfoxide (MSO).

Other non-conventional amino acids are listed in Table 6.

TABLE 6 Non-conventional amino acid Code Non-conventional amino acidCode α-aminobutyric acid Abu L-N-methylalanine Nmalaα-amino-α-methylbutyrate Mgabu L-N-methylarginine Nmargaminocyclopropane- Cpro L-N-methylasparagine Nmasn carboxylateL-N-methylaspartic acid Nmasp aminoisobutyric acid AibL-N-methylcysteine Nmcys aminonorbornyl- Norb L-N-methylglutamine Nmgincarboxylate L-N-methylglutamic acid Nmglu cyclohexylalanine ChexaL-N-methylhistidine Nmhis cyclopentylalanine Cpen L-N-methylisolleucineNmile D-alanine Dal L-N-methylleucine Nmleu D-arginine DargL-N-methyllysine Nmlys D-aspartic acid Dasp L-N-methylmethionine NmmetD-cysteine Dcys L-N-methylnorleucine Nmnle D-glutamine DglnL-N-methylnorvaline Nmnva D-glutamic acid Dglu L-N-methylornithine NmornD-histidine Dhis L-N-methylphenylalanine Nmphe D-isoleucine DileL-N-methylproline Nmpro D-leucine Dleu L-N-methylserine Nmser D-lysineDlys L-N-methylthreonine Nmthr D-methionine Dmet L-N-methyltryptophanNmtrp D-ornithine Dorn L-N-methyltyrosine Nmtyr D-phenylalanine DpheL-N-methylvaline Nmval D-proline Dpro L-N-methylethylglycine NmetgD-serine Dser L-N-methyl-t-butylglycine Nmtbug D-threonine DthrL-norleucine Nle D-tryptophan Dtrp L-norvaline Nva D-tyrosine Dtyrα-methyl-aminoisobutyrate Maib D-valine Dval α-methyl-γ-aminobutyrateMgabu D-α-methylalanine Dmala α-methylcyclohexylalanine MchexaD-α-methylarginine Dmarg α-methylcyclopentylalanine McpenD-α-methylasparagine Dmasn α-methyl-α-napthylalanine ManapD-α-methylaspartate Dmasp α-methylpenicillamine Mpen D-α-methylcysteineDmcys N-(4-aminobutyl)glycine Nglu D-α-methylglutamine DmglnN-(2-aminoethyl)glycine Naeg D-α-methylhistidine DmhisN-(3-aminopropyl)glycine Norn D-α-methylisoleucine DmileN-amino-α-methylbutyrate Nmaabu D-α-methylleucine Dmleu α-napthylalanineAnap D-α-methyllysine Dmlys N-benzylglycine Nphe D-α-methylmethionineDmmet N-(2-carbamylethyl)glycine Ngln D-α-methylornithine DmornN-(carbamylmethyl)glycine Nasn D-α-methylphenylalanine DmpheN-(2-carboxyethyl)glycine Nglu D-α-methylproline DmproN-(carboxymethyl)glycine Nasp D-α-methylserine Dmser N-cyclobutylglycineNcbut D-α-methylthreonine Dmthr N-cycloheptylglycine NchepD-α-methyltryptophan Dmtrp N-cyclohexylglycine Nchex D-α-methyltyrosineDmty N-cyclodecylglycine Ncdec D-α-methylvaline DmvalN-cyclododeclglycine Ncdod D-α-methylalnine Dnmala N-cyclooctylglycineNcoct D-α-methylarginine Dnmarg N-cyclopropylglycine NcproD-α-methylasparagine Dnmasn N-cycloundecylglycine NcundD-α-methylasparatate Dnmasp N-(2,2-diphenylethyl)glycine NbhmD-α-methylcysteine Dnmcys N-(3,3-diphenylpropyl)glycine NbheD-N-methylleucine Dnmleu N-(3-indolylyethyl) glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvaD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomo phenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetD-N-methylglutamine Dnmgln N-(3-guanidinopropyl)glycine NargD-N-methylglutamate Dnmglu N-(1-hydroxyethyl)glycine NthrD-N-methylhistidine Dnmhis N-(hydroxyethyl)glycine NserD-N-methylisoleucine Dnmile N-(imidazolylethyl)glycine NhisD-N-methylleucine Dnmleu N-(3-indolylyethyl)glycine NhtrpD-N-methyllysine Dnmlys N-methyl-γ-aminobutyrate NmgabuN-methylcyclohexylalanine Nmchexa D-N-methylmethionine DnmmetD-N-methylornithine Dnmorn N-methylcyclopentylalanine NmcpenN-methylglycine Nala D-N-methylphenylalanine DnmpheN-methylaminoisobutyrate Nmaib D-N-methylproline DnmproN-(1-methylpropyl)glycine Nile D-N-methylserine DnmserN-(2-methylpropyl)glycine Nleu D-N-methylthreonine DnmthrD-N-methyltryptophan Dnmtrp N-(1-methylethyl)glycine NvalD-N-methyltyrosine Dnmtyr N-methyla-napthylalanine NmanapD-N-methylvaline Dnmval N-methylpenicillamine Nmpen γ-aminobutyric acidGabu N-(p-hydroxyphenyl)glycine Nhtyr L-t-butylglycine TbugN-(thiomethyl)glycine Ncys L-ethylglycine Etg penicillamine PenL-homophenylalanine Hphe L-α-methylalanine Mala L-α-methylarginine MargL-α-methylasparagine Masn L-α-methylaspartate MaspL-α-methyl-t-butylglycine Mtbug L-α-methylcysteine McysL-methylethylglycine Metg L-α-methylglutamine Mgln L-α-methylglutamateMglu L-α-methylhistidine Mhis L-α-methylhomophenylalanine MhpheL-α-methylisoleucine Mile N-(2-methylthioethyl)glycine NmetL-α-methylleucine Mleu L-α-methyllysine Mlys L-α-methylmethionine MmetL-α-methylnorleucine Mnle L-α-methylnorvaline Mnva L-α-methylornithineMorn L-α-methylphenylalanine Mphe L-α-methylproline MproL-α-methylserine mser L-α-methylthreonine Mthr L-α-methylvaline MtrpL-α-methyltyrosine Mtyr L-α-methylleucine MvalL-N-methylhomophenylalanine Nmhphe Nnbhm N-(N-(2,2-diphenylethyl)N-(N-(3,3-diphenylpropyl) carbamylmethyl-glycine Nnbhmcarbamylmethyl(1)glycine Nnbhe1-carboxy-1-(2,2-diphenylethylamino)cyclopropane Nmbc

Modifications Fusion Proteins

A fusion protein may be prepared from a peptide according to the presentinvention by fusion with a portion of an immunoglobulin comprising aconstant region of an immunoglobulin.

More preferably, the portion of the immunoglobulin comprises a heavychain constant region which is optionally and more preferably a humanheavy chain constant region. The heavy chain constant region is mostpreferably an IgG heavy chain constant region, and optionally and mostpreferably is an Fc chain, most preferably an IgG Fc fragment thatcomprises CH2 and CH3 domains. Although any IgG subtype may optionallybe used, the IgG1 subtype is preferred. The Fc chain may optionally be aknown or “wild type” Fc chain, or alternatively may be mutated.Non-limiting, illustrative, exemplary types of mutations are describedin US Patent Application No. 20060034852, published on Feb. 16 2006,hereby incorporated by reference as if fully set forth herein. The term“Fc chain” also optionally comprises any type of Fc fragment.

Several of the specific amino acid residues that are important forantibody constant region-mediated activity in the IgG subclass have beenidentified. Inclusion, substitution or exclusion of these specific aminoacids therefore allows for inclusion or exclusion of specificimmunoglobulin constant region-mediated activity. Furthermore, specificchanges may result in aglycosylation for example and/or other desiredchanges to the Fc chain. At least some changes may optionally be made toblock a function of Fc which is considered to be undesirable, such as anundesirable immune system effect, as described in greater detail below.

Non-limiting, illustrative examples of mutations to Fc which may be madeto modulate the activity of the fusion protein include the followingchanges (given with regard to the Fc sequence nomenclature as given byKabat, from Kabat E A et al: Sequences of Proteins of

Immunological Interest. US Department of Health and Human Services, NIH,1991): 220C->S; 233-238 ELLGGP->EAEGAP; 265D->A, preferably incombination with 434N->A; 297N->A (for example to blockN-glycosylation); 318-322 EYKCK->AYACA; 330-331AP->SS; or a combinationthereof (see for example M. Clark, “Chemical Immunol and AntibodyEngineering”, pp 1-31 for a description of these mutations and theireffect). The construct for the Fc chain which features the above changesoptionally and preferably comprises a combination of the hinge regionwith the CH2 and CH3 domains.

The above mutations may optionally be implemented to enhance desiredproperties or alternatively to block non-desired properties. Forexample, aglycosylation of antibodies was shown to maintain the desiredbinding functionality while blocking depletion of T-cells or triggeringcytokine release, which may optionally be undesired functions (see M.Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Substitution of 331 proline for serine may block the ability to activatecomplement, which may optionally be considered an undesired function(see M. Clark, “Chemical Immunol and Antibody Engineering”, pp 1-31).Changing 330 alanine to serine in combination with this change may alsoenhance the desired effect of blocking the ability to activatecomplement.

Residues 235 and 237 were shown to be involved in antibody-dependentcell-mediated cytotoxicity (ADCC), such that changing the block ofresidues from 233-238 as described may also block such activity if ADCCis considered to be an undesirable function.

Residue 220 is normally a cysteine for Fc from IgG1, which is the siteat which the heavy chain forms a covalent linkage with the light chain.Optionally, this residue may be changed to a serine, to avoid any typeof covalent linkage (see M. Clark, “Chemical Immunol and AntibodyEngineering”, pp 1-31).

The above changes to residues 265 and 434 may optionally be implementedto reduce or block binding to the Fc receptor, which may optionallyblock undesired functionality of Fc related to its immune systemfunctions (see “Binding site on Human IgG1 for Fc Receptors”, Shields etal. vol 276, pp 6591-6604, 2001).

The above changes are intended as illustrations only of optional changesand are not meant to be limiting in any way. Furthermore, the aboveexplanation is provided for descriptive purposes only, without wishingto be bound by a single hypothesis.

Addition of Groups

If a peptide according to the present invention is a linear molecule, itis possible to place various functional groups at various points on thelinear molecule which are susceptible to or suitable for chemicalmodification. Functional groups can be added to the termini of linearforms of the peptide. In some embodiments, the functional groups improvethe activity of the peptide with regard to one or more characteristics,including but not limited to, improvement in stability, penetration(through cellular membranes and/or tissue barriers), tissuelocalization, efficacy, decreased clearance, decreased toxicity,improved selectivity, improved resistance to expulsion by cellularpumps, and the like. For convenience sake and without wishing to belimiting, the free N-terminus of one of the sequences contained in thecompositions of the invention will be termed as the N-terminus of thecomposition, and the free C-terminal of the sequence will be consideredas the C-terminus of the composition. Either the C-terminus or theN-terminus of the sequences, or both, can be linked to a carboxylic acidfunctional groups or an amine functional group, respectively.

Non-limiting examples of suitable functional groups are described inGreen and Wuts, “Protecting Groups in Organic Synthesis”, John Wiley andSons, Chapters 5 and 7, 1991, the teachings of which are incorporatedherein by reference. Preferred protecting groups are those thatfacilitate transport of the active ingredient attached thereto into acell, for example, by reducing the hydrophilicity and increasing thelipophilicity of the active ingredient, these being an example for “amoiety for transport across cellular membranes”.

These moieties can optionally and preferably be cleaved in vivo, eitherby hydrolysis or enzymatically, inside the cell. (Ditter et al., J.Pharm. Sci. 57:783 (1968); Ditter et al., J. Pharm. Sci. 57:828 (1968);Ditter et al., J. Pharm. Sci. 58:557 (1969); King et al., Biochemistry26:2294 (1987); Lindberg et al., Drug Metabolism and Disposition 17:311(1989); and Tunek et al., Biochem. Pharm. 37:3867 (1988), Anderson etal., Arch. Biochem. Biophys. 239:538 (1985) and Singhal et al., FASEB J.1:220 (1987)). Hydroxyl protecting groups include esters, carbonates andcarbamate protecting groups. Amine protecting groups include alkoxy andaryloxy carbonyl groups, as described above for N-terminal protectinggroups. Carboxylic acid protecting groups include aliphatic, benzylicand aryl esters, as described above for C-terminal protecting groups. Inone embodiment, the carboxylic acid group in the side chain of one ormore glutamic acid or aspartic acid residue in a composition of thepresent invention is protected, preferably with a methyl, ethyl, benzylor substituted benzyl ester, more preferably as a benzyl ester.

Non-limiting, illustrative examples of N-terminal protecting groupsinclude acyl groups (—CO—R1) and alkoxy carbonyl or aryloxy carbonylgroups (—CO—O—R1), wherein R1 is an aliphatic, substituted aliphatic,benzyl, substituted benzyl, aromatic or a substituted aromatic group.Specific examples of acyl groups include but are not limited to acetyl,(ethyl)-CO—, n-propyl-CO—, iso-propyl-CO—, n-butyl-CO—, sec-butyl-CO—,t-butyl-CO—, hexyl, lauroyl, palmitoyl, myristoyl, stearyl, oleoylphenyl-CO—, substituted phenyl-CO—, benzyl-CO— and (substitutedbenzyl)-CO—. Examples of alkoxy carbonyl and aryloxy carbonyl groupsinclude CH3-O—CO—, (ethyl)-O—CO—, n-propyl-O—CO—, iso-propyl-O—CO—,n-butyl-O—CO—, sec-butyl-O—CO—, t-butyl-O—CO—, phenyl-O—CO—, substitutedphenyl-O—CO— and benzyl-O—CO—, (substituted benzyl)-O—OC—, Adamantan,naphtalen, myristoleyl, toluen, biphenyl, cinnamoyl, nitrobenzoy,toluoyl, furoyl, benzoyl, cyclohexane, norbornane, or Z-caproic. Inorder to facilitate the N-acylation, one to four glycine residues can bepresent in the N-terminus of the molecule.

The carboxyl group at the C-terminus of the compound can be protected,for example, by a group including but not limited to an amide (i.e., thehydroxyl group at the C-terminus is replaced with —NH₂, —NHR₂ and—NR₂R₃) or ester (i.e. the hydroxyl group at the C-terminus is replacedwith —OR₂). R₂ and R₃ are optionally independently an aliphatic,substituted aliphatic, benzyl, substituted benzyl, aryl or a substitutedaryl group. In addition, taken together with the nitrogen atom, R₂ andR₃ can optionally form a C4 to C8 heterocyclic ring with from about 0-2additional heteroatoms such as nitrogen, oxygen or sulfur. Non-limitingsuitable examples of suitable heterocyclic rings include piperidinyl,pyrrolidinyl, morpholino, thiomorpholino or piperazinyl. Examples ofC-terminal protecting groups include but are not limited to —NH₂,—NHCH₃, —N(CH₃)₂, —NH(ethyl), —N(ethyl)₂, —N(methyl) (ethyl),—NH(benzyl), —N(C1-C4 alkyl)(benzyl), —NH(phenyl), —N(C1-C4 alkyl)(phenyl), —OCH₃, —O-(ethyl), —O-(n-propyl), —O-(n-butyl),—O-(iso-propyl), —O-(sec-butyl), —O-(t-butyl), —O-benzyl and —O-phenyl.

Substitution by Peptidomimetic Moieties

A “peptidomimetic organic moiety” can optionally be substituted foramino acid residues in the composition of this invention both asconservative and as non-conservative substitutions. These moieties arealso termed “non-natural amino acids” and may optionally replace aminoacid residues, amino acids or act as spacer groups within the peptidesin lieu of deleted amino acids. The peptidomimetic organic moietiesoptionally and preferably have steric, electronic or configurationalproperties similar to the replaced amino acid and such peptidomimeticsare used to replace amino acids in the essential positions, and areconsidered conservative substitutions. However such similarities are notnecessarily required. According to preferred embodiments of the presentinvention, one or more peptidomimetics are selected such that thecomposition at least substantially retains its physiological activity ascompared to the native peptide protein according to the presentinvention.

Peptidomimetics may optionally be used to inhibit degradation of thepeptides by enzymatic or other degradative processes. Thepeptidomimetics can optionally and preferably be produced by organicsynthetic techniques. Non-limiting examples of suitable peptidomimeticsinclude D amino acids of the corresponding L amino acids, tetrazol(Zabrocki et al., J. Am. Chem. Soc. 110:5875-5880 (1988)); isosteres ofamide bonds (Jones et al., Tetrahedron Lett. 29: 3853-3856 (1988));LL-3-amino-2-propenidone-6-carboxylic acid (LL-Acp) (Kemp et al., J.Org. Chem. 50:5834-5838 (1985)). Similar analogs are shown in Kemp etal., Tetrahedron Lett. 29:5081-5082 (1988) as well as Kemp et al.,Tetrahedron Lett. 29:5057-5060 (1988), Kemp et al., Tetrahedron Lett.29:4935-4938 (1988) and Kemp et al., J. Org. Chem. 54:109-115 (1987).Other suitable but exemplary peptidomimetics are shown in Nagai andSato, Tetrahedron Lett. 26:647-650 (1985); Di Maio et al., J. Chem. Soc.Perkin Trans., 1687 (1985); Kahn et al., Tetrahedron Lett. 30:2317(1989); Olson et al., J. Am. Chem. Soc. 112:323-333 (1990); Garvey etal., J. Org. Chem. 56:436 (1990). Further suitable exemplarypeptidomimetics includehydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al., J.Takeda Res. Labs 43:53-76 (1989));1,2,3,4-tetrahydro-isoquinoline-3-carboxylate (Kazmierski et al., J. Am.Chem. Soc. 133:2275-2283 (1991)); histidine isoquinolone carboxylic acid(HIC) (Zechel et al., Int. J. Pep. Protein Res. 43 (1991));(2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine,(2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine(Kazmierski and Hruby, Tetrahedron Lett. (1991)).

Exemplary, illustrative but non-limiting non-natural amino acids includebeta-amino acids (beta3 and beta2), homo-amino acids, cyclic aminoacids, aromatic amino acids, Pro and Pyr derivatives, 3-substitutedAlanine derivatives, Glycine derivatives, ring-substituted Phe and TyrDerivatives, linear core amino acids or diamino acids. They areavailable from a variety of suppliers, such as Sigma-Aldrich (USA) forexample.

Chemical Modifications

In the present invention any part of a peptide may optionally bechemically modified, i.e. changed by addition of functional groups. Forexample the side amino acid residues appearing in the native sequencemay optionally be modified, although as described below alternativelyother part(s) of the protein may optionally be modified, in addition toor in place of the side amino acid residues. The modification mayoptionally be performed during synthesis of the molecule if a chemicalsynthetic process is followed, for example by adding a chemicallymodified amino acid. However, chemical modification of an amino acidwhen it is already present in the molecule (“in situ” modification) isalso possible.

The amino acid of any of the sequence regions of the molecule canoptionally be modified according to any one of the following exemplarytypes of modification (in the peptide conceptually viewed as “chemicallymodified”). Non-limiting exemplary types of modification includecarboxymethylation, acylation, phosphorylation, glycosylation or fattyacylation. Ether bonds can optionally be used to join the serine orthreonine hydroxyl to the hydroxyl of a sugar. Amide bonds canoptionally be used to join the glutamate or aspartate carboxyl groups toan amino group on a sugar (Garg and Jeanloz, Advances in CarbohydrateChemistry and Biochemistry, Vol. 43, Academic Press (1985); Kunz, Ang.Chem. Int. Ed. English 26:294-308 (1987)). Acetal and ketal bonds canalso optionally be formed between amino acids and carbohydrates. Fattyacid acyl derivatives can optionally be made, for example, by acylationof a free amino group (e.g., lysine) (Toth et al., Peptides: Chemistry,Structure and Biology, Rivier and Marshal, eds., ESCOM Publ., Leiden,1078-1079 (1990)).

As used herein the term “chemical modification”, when referring to aprotein or peptide according to the present invention, refers to aprotein or peptide where at least one of its amino acid residues ismodified either by natural processes, such as processing or otherpost-translational modifications, or by chemical modification techniqueswhich are well known in the art. Examples of the numerous knownmodifications typically include, but are not limited to:

acetylation, acylation, amidation, ADP-ribosylation, glycosylation, GPIanchor formation, covalent attachment of a lipid or lipid derivative,methylation, myristylation, pegylation, prenylation, phosphorylation,ubiquitination, or any similar process.

Other types of modifications optionally include the addition of acycloalkane moiety to a biological molecule, such as a protein, asdescribed in PCT Application No. WO 2006/050262, hereby incorporated byreference as if fully set forth herein. These moieties are designed foruse with biomolecules and may optionally be used to impart variousproperties to proteins.

Furthermore, optionally any point on a protein may be modified. Forexample, pegylation of a glycosylation moiety on a protein mayoptionally be performed, as described in PCT Application No. WO2006/050247, hereby incorporated by reference as if fully set forthherein. One or more polyethylene glycol (PEG) groups may optionally beadded to O-linked and/or N-linked glycosylation. The PEG group mayoptionally be branched or linear. Optionally any type of water-solublepolymer may be attached to a glycosylation site on a protein through aglycosyl linker.

Covalent modifications of the peptides of the present invention areincluded within the scope of this invention. Other types of covalentmodifications of the peptides are introduced into the molecule byreacting targeted amino acid residues with an organic derivatizing agentthat is capable of reacting with selected side chains or the N- orC-terminal residues.

Cysteinyl residues most commonly are reacted with α-haloacetates (andcorresponding amines), such as chloroacetic acid or chloroacetamide, togive carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residuesalso are derivatized by reaction with bromotrifluoroacetone,α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate,N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyldisulfide, p-chloromercuribenzoate, 2-chloromercuri-4-nitrophenol, orchloro-7-nitrobenzo-2-oxa-1,3-diazole.

Histidyl residues are derivatized by reaction with diethylpyrocarbonateat pH 5.5-7.0 because this agent is relatively specific for the histidylside chain. Para-bromophenacyl bromide also is useful; the reaction ispreferably performed in 0.1M sodium cacodylate at pH 6.0.

Lysinyl and amino-terminal residues are reacted with succinic or othercarboxylic acid anhydrides. Derivatization with these agents has theeffect of reversing the charge of the lysinyl residues. Other suitablereagents for derivatizing α-amino-containing residues includeimidoesters such as methyl picolinimidate, pyridoxal phosphate,pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid,O-methylisourea, 2,4-pentanedione, and transaminase-catalyzed reactionwith glyoxylate.

Arginyl residues are modified by reaction with one or severalconventional reagents, among them phenylglyoxal, 2,3-butanedione,1,2-cyclohexanedione, and ninhydrin. Derivatization of arginine residuesrequires that the reaction be performed in alkaline conditions becauseof the high pKa of the guanidine functional group. Furthermore, thesereagents may react with the groups of lysine as well as the arginineepsilon-amino group.

The specific modification of tyrosyl residues may be made, withparticular interest in introducing spectral labels into tyrosyl residuesby reaction with aromatic diazonium compounds or tetranitromethane. Mostcommonly, N-acetylimidizole and tetranitromethane are used to form0-acetyl tyrosyl species and 3-nitro derivatives, respectively. Tyrosylresidues are iodinated using 125 I or 131 I to prepare labeled proteinsfor use in radioimmunoassay, the chloramine T method described abovebeing suitable.

Carboxyl side groups (aspartyl or glutamyl) are selectively modified byreaction with carbodiimides (R—N═C═N—R′), where R and R′ are differentalkyl groups, such as 1-cyclohexyl-3-(2-morpholinyl-4-ethyl)carbodiimideor 1-ethyl-3-(4-azonia-4,4-dimethylpentyl) carbodiimide. Furthermore,aspartyl and glutamyl residues are converted to asparaginyl andglutaminyl residues by reaction with ammonium ions.

Derivatization with bifunctional agents is useful for crosslinking CHFto a water-insoluble support matrix or surface for use in the method forpurifying anti-CHF antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis(diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),and bifunctional maleimides such as bis-N-maleimido-1,8-octane.Derivatizing agents such asmethyl-3-[(p-azidophenyl)dithio]propioimidate yield photoactivatableintermediates that are capable of forming crosslinks in the presence oflight. Alternatively, reactive water-insoluble matrices such as cyanogenbromide-activated carbohydrates and the reactive substrates described inU.S. Pat. Nos. 3,969,287; 3,691,016; 4,195,128; 4,247,642; 4,229,537;and 4,330,440 are employed for protein immobilization.

Glutaminyl and asparaginyl residues are frequently deamidated to thecorresponding glutamyl and aspartyl residues, respectively. Theseresidues are deamidated under neutral or basic conditions. Thedeamidated form of these residues falls within the scope of thisinvention.

Other modifications include hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the α-amino groups of lysine, arginine, and histidineside chains (T. E. Creighton, Proteins: Structure and MolecularProperties, W. H. Freeman & Co., San Francisco, pp. 79-86 [1983]),acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

Altered Glycosylation

Peptides of the invention may be modified to have an alteredglycosylation pattern (i.e., altered from the original or nativeglycosylation pattern). As used herein, “altered” means having one ormore carbohydrate moieties deleted, and/or having at least oneglycosylation site added to the original protein.

Glycosylation of proteins is typically either N-linked or O-linked.N-linked refers to the attachment of the carbohydrate moiety to the sidechain of an asparagine residue. The tripeptide sequences,asparagine-X-serine and asparagine-X-threonine, where X is any aminoacid except proline, are the recognition sequences for enzymaticattachment of the carbohydrate moiety to the asparagine side chain.Thus, the presence of either of these tripeptide sequences in apolypeptide creates a potential glycosylation site. O-linkedglycosylation refers to the attachment of one of the sugarsN-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, mostcommonly serine or threonine, although 5-hydroxyproline or5-hydroxylysine may also be used.

Addition of glycosylation sites to peptides of the invention isconveniently accomplished by altering the amino acid sequence of theprotein such that it contains one or more of the above-describedtripeptide sequences (for N-linked glycosylation sites). The alterationmay also be made by the addition of, or substitution by, one or moreserine or threonine residues in the sequence of the original protein(for O-linked glycosylation sites). The protein's amino acid sequencemay also be altered by introducing changes at the DNA level.

Another means of increasing the number of carbohydrate moieties onproteins is by chemical or enzymatic coupling of glycosides to the aminoacid residues of the protein. Depending on the coupling mode used, thesugars may be attached to (a) arginine and histidine, (b) free carboxylgroups, (c) free sulfhydryl groups such as those of cysteine, (d) freehydroxyl groups such as those of serine, threonine, or hydroxyproline,(e) aromatic residues such as those of phenylalanine, tyrosine, ortryptophan, or (f) the amide group of glutamine. These methods aredescribed in WO 87/05330, and in Aplin and Wriston, CRC Crit. Rev.Biochem., 22: 259-306 (1981).

Removal of any carbohydrate moieties present on peptides of theinvention may be accomplished chemically or enzymatically. Chemicaldeglycosylation requires exposure of the protein totrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), leaving the amino acidsequence intact.

Chemical deglycosylation is described by Hakimuddin et al., Arch.Biochem. Biophys., 259: 52 (1987); and Edge et al., Anal. Biochem., 118:131 (1981). Enzymatic cleavage of carbohydrate moieties on proteins canbe achieved by the use of a variety of endo- and exo-glycosidases asdescribed by Thotakura et al., Meth. Enzymol., 138: 350 (1987).

Antibodies to Bioactive Peptides

The invention also includes an antibody to a bioactive peptide disclosedherein, or a fragment of the bioactive peptide. In some embodiments, thebioactive peptide is a GPCR ligand.

“Antibody” refers to a polypeptide ligand substantially encoded by animmunoglobulin gene or immunoglobulin genes, or fragments thereof, whichspecifically binds and recognizes an epitope (e.g., an antigen). Theantibody can be provided as, e.g., an intact immunoglobulin or asfragment, e.g., a fragment produced by digestion with variouspeptidases. This includes, e.g., Fab′ and F(ab)′₂ Fv (defined as agenetically engineered fragment containing the variable region of thelight chain and the variable region of the heavy chain expressed as twochains); and (5) Single chain antibody (“SCA”), a genetically engineeredmolecule containing the variable region of the light chain and thevariable region of the heavy chain, linked by a suitable polypeptidelinker as a genetically fused single chain molecule. The term“antibody,” as used herein, also includes antibody fragments eitherproduced by the modification of whole antibodies or those synthesized denovo using recombinant DNA methodologies. It includes polyclonalantibodies, monoclonal antibodies, chimeric antibodies, humanizedantibodies, or single chain antibodies. “Fc” portion of an antibodyrefers to that portion of an immunoglobulin heavy chain that comprisesone or more heavy chain constant region domains, CH1, CH2 and CH3, butdoes not include the heavy chain variable region.

Antibodies are raised against, e.g., an epitope in a peptide of FormulaI. In some embodiments, anti-GPCR peptide ligand antibodies are raisedagainst

Peptide P59-S-Amide (Amide): (SEQ ID NO: 1) AYAAFSV-Amide; PeptideP59-SG (free acid Gly) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide)(SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid)(SEQ ID NO: 4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide)(SEQ ID NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide Peptide P59C13V (free acid)(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; Peptide P74-Amide (Amide): (SEQID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74 (free acid)(SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V (amide)(SEQ ID NO: 9) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG.

Methods of producing polyclonal and monoclonal antibodies as well asfragments thereof are well known in the art (See for example, Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988, incorporated herein by reference).

Antibody fragments can be prepared by proteolytic hydrolysis of theantibody or by expression in E. coli or mammalian cells (e.g. Chinesehamster ovary cell culture or other protein expression systems) of DNAencoding the fragment. Antibody fragments can be obtained by pepsin orpapain digestion of whole antibodies by conventional methods.

The bioactive peptide antibody can additionally be provided as a peptidecoding corresponding a single complementarity-determining region (CDR).CDR peptides (“minimal recognition units”) can be obtained byconstructing genes encoding the CDR of an antibody of interest. Suchgenes are prepared, for example, by using the polymerase chain reactionto synthesize the variable region from RNA of antibody-producing cells.See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

Humanized forms of non-human (e.g., murine) antibodies are chimericmolecules of immunoglobulins, immunoglobulin chains or fragments thereof(such as Fv, Fab, Fab′, F(ab′) or other antigen-binding subsequences ofantibodies) which contain minimal sequence derived from non-humanimmunoglobulin. Humanized antibodies include human immunoglobulins(recipient antibody) in which residues from a complementary determiningregion (CDR) of the recipient are replaced by residues from a CDR of anon-human species (donor antibody) such as mouse, rat or rabbit havingthe desired specificity, affinity and capacity. In some instances, Fvframework residues of the human immunoglobulin are replaced bycorresponding non-human residues. Humanized antibodies may also compriseresidues which are found neither in the recipient antibody nor in theimported CDR or framework sequences. In general, the humanized antibodywill comprise substantially all of at least one, and typically two,variable domains, in which all or substantially all of the CDR regionscorrespond to those of a non-human immunoglobulin and all orsubstantially all of the FR regions are those of a human immunoglobulinconsensus sequence. The humanized antibody optimally also will compriseat least a portion of an immunoglobulin constant region (Fc), typicallythat of a human immunoglobulin [Jones et al., Nature, 321:522-525(1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr.Op. Struct. Biol., 2:593-596 (1992)].

Methods for humanizing non-human antibodies are well known in the art.Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as import residues, which aretypically taken from an import variable domain. Humanization can beessentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such humanized antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

Human antibodies can also be produced using various techniques known inthe art, including phage display libraries [Hoogenboom and Winter, J.Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581(1991)]. The techniques of Cole et al. and Boerner et al. are alsoavailable for the preparation of human monoclonal antibodies (Cole etal., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introduction of human immunoglobulinloci into transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10: 779-783(1992); Lonberg et al., Nature 368: 856-859 (1994); Morrison, Nature 368812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14: 826 (1996); and Lonberg and Huszar,Intern. Rev. Immunol. 13, 65-93 (1995).

The antibody preferably binds specifically (or selectively) to a GPCRpeptide ligand. The phrase “specifically (or selectively) binds” to anantibody or “specifically (or selectively) immunoreactive with,” whenreferring to a protein or peptide, refers to a binding reaction that isdeterminative of the presence of the protein in a heterogeneouspopulation of proteins and other biologics. Thus, under designatedimmunoassay conditions, the specified antibodies bind to a particularprotein at least two times the background and do not substantially bindin a significant amount to other proteins present in the sample.Specific binding to an antibody under such conditions may require anantibody that is selected for its specificity for a particular protein.A variety of immunoassay formats may be used to select antibodiesspecifically immunoreactive with a particular protein. For example,solid-phase ELISA immunoassays are routinely used to select antibodiesspecifically immunoreactive with a protein (see, e.g., Harlow & Lane,Antibodies, A Laboratory Manual (1988), for a description of immunoassayformats and conditions that can be used to determine specificimmunoreactivity). Typically a specific or selective reaction will be atleast twice background signal or noise and more typically more than 10to 100 times background.

If desired, the antibody can be provided conjugated or coupled to adetectable label, a radioactive label, an enzyme, a fluorescent label, aluminescent label, a bioluminescent label, or a therapeutic agent.

Methods of Treatment

According to an additional aspect of the present invention there isprovided a method of treating disease, disorder or condition, asdescribed hereinabove, in a subject.

The subject according to the present invention is a mammal, preferably ahuman which is diagnosed with one of the disease, disorder or conditionsdescribed hereinabove, or alternatively is predisposed to at least onetype of disease, disorder or conditions described hereinabove.

As used herein the term “treating” refers to preventing, curing,reversing, attenuating, alleviating, minimizing, suppressing or haltingthe deleterious effects of the above-described diseases, disorders orconditions.

Treating, according to the present invention, can be effected byspecifically upregulating the expression of at least one of thepolypeptides of the present invention in the subject.

Optionally, upregulation may be effected by administering to the subjectat least one of the polypeptides of the present invention (e.g.,recombinant or synthetic) or an active portion thereof, as describedherein. The polypeptide or peptide may optionally be administered in aspart of a pharmaceutical composition, described in more detail below.

It will be appreciated that treatment of the above-described diseasesaccording to the present invention may be combined with other treatmentmethods known in the art (i.e., combination therapy). Thus, treatment ofmalignancies using the agents of the present invention may be combinedwith, for example, radiation therapy, antibody therapy and/orchemotherapy, surgery or in combination therapy with other biologicalagents, conventional drugs and/or with any other anti-cancer agent, suchas immunosuppressants or cytotoxic drugs for cancer, and or incombination with therapeutic agents targeting complement regulatoryproteins (CRPs). Each one of the above mentioned compounds orpharmaceutical composition of the invention may also be administered inconjunction with other compounds. For example, the combination therapycan include the compound of the present invention combined with at leastone other therapeutic or immune modulatory agent, including, but notlimited to, chemotherapeutic agents such as cytotoxic and cytostaticagents, immunological modifiers such as interferons and interleukins,growth hormones or other cytokines, folic acid, vitamins, minerals,aromatase inhibitors, RNAi, Histone Deacetylase Inhibitors, proteasomeinhibitors, and so forth.

Alternatively or additionally, an upregulating method may optionally beeffected by specifically upregulating the amount (optionally expression)in the subject of at least one of the polypeptides of the presentinvention or active portions thereof.

Upregulating expression of the therapeutic peptides of the presentinvention may be effected via the administration of at least one of theexogenous polynucleotide sequences of the present invention, ligatedinto a nucleic acid expression construct designed for expression ofcoding sequences in eukaryotic cells (e.g., mammalian cells).Accordingly, the exogenous polynucleotide sequence may be a DNA or RNAsequence encoding the peptides of the present invention or activeportions thereof.

It will be appreciated that the nucleic acid construct can beadministered to the individual employing any suitable mode ofadministration including in vivo gene therapy (e.g., using viraltransformation as described hereinabove). Alternatively, the nucleicacid construct is introduced into a suitable cell via an appropriategene delivery vehicle/method (transfection, transduction, homologousrecombination, etc.) and an expression system as needed and then themodified cells are expanded in culture and returned to the individual(i.e., ex-vivo gene therapy).

Such cells (i.e., which are transfected with the nucleic acid constructof the present invention) can be any suitable cells, such as kidney,bone marrow, keratinocyte, lymphocyte, adult stem cells, cord bloodcells, embryonic stem cells which are derived from the individual andare transfected ex vivo with an expression vector containing thepolynucleotide designed to express the polypeptide of the presentinvention as described hereinabove.

Administration of the ex vivo transfected cells of the present inventioncan be effected using any suitable route such as intravenous, intraperitoneal, intra kidney, intra gastrointestinal track, subcutaneous,transcutaneous, intramuscular, intracutaneous, intrathecal, epidural andrectal. According to presently preferred embodiments, the ex vivotransfected cells of the present invention are introduced to theindividual using intravenous, intra kidney, intra gastrointestinal trackand/or intra peritoneal administrations.

The ex vivo transfected cells of the present invention can be derivedfrom either autologous sources such as self bone marrow cells or fromallogeneic sources such as bone marrow or other cells derived fromnon-autologous sources. Since non-autologous cells are likely to inducean immune reaction when administered to the body several approaches havebeen developed to reduce the likelihood of rejection of non-autologouscells. These include either suppressing the recipient immune system orencapsulating the non-autologous cells or tissues in immunoisolating,semipermeable membranes before transplantation.

Encapsulation techniques are generally classified as microencapsulation,involving small spherical vehicles and macroencapsulation, involvinglarger flat-sheet and hollow-fiber membranes (Uludag, H. et al.Technology of mammalian cell encapsulation. Adv Drug Deliv Rev. 2000;42: 29-64).

Methods of preparing microcapsules are known in the arts and include forexample those disclosed by Lu M Z, et al., Cell encapsulation withalginate and alpha-phenoxycinnamylidene-acetylated poly(allylamine).Biotechnol Bioeng. 2000, 70: 479-83, Chang T M and Prakash S. Proceduresfor microencapsulation of enzymes, cells and genetically engineeredmicroorganisms. Mol Biotechnol. 2001, 17: 249-60, and Lu M Z, et al., Anovel cell encapsulation method using photosensitive poly (allylaminealpha-cyanocinnamylideneacetate). J Microencapsul. 2000, 17: 245-51.

For example, microcapsules are prepared by complexing modified collagenwith a ter-polymer shell of 2-hydroxyethyl methylacrylate (HEMA),methacrylic acid (MAA) and methyl methacrylate (MMA), resulting in acapsule thickness of 2-5 μm. Such microcapsules can be furtherencapsulated with additional 2-5 μm ter-polymer shells in order toimpart a negatively charged smooth surface and to minimize plasmaprotein absorption (Chia, S. M. et al. Multi-layered microcapsules forcell encapsulation Biomaterials. 2002 23: 849-56).

Other microcapsules are based on alginate, a marine polysaccharide(Sambanis, A. Encapsulated islets in diabetes treatment. DiabetesThechnol. Ther. 2003, 5: 665-8) or its derivatives. For example,microcapsules can be prepared by the polyelectrolyte complexationbetween the polyanions sodium alginate and sodium cellulose sulphatewith the polycation poly(methylene-co-guanidine) hydrochloride in thepresence of calcium chloride.

Pharmaceutical Compositions and Delivery Thereof

The bioactive peptide ligand is typically provided in a pharmaceuticallyacceptable carrier suitable for administering the pharmaceuticalcomposition to a human patient. As would be appreciated by one of skillin this art, the carriers may be chosen based on the route ofadministration as described below, the location of the target issue, thedrug being delivered, the time course of delivery of the drug, etc.

The term “pharmaceutically acceptable carrier” means a non-toxic, inertsolid, semi-solid or liquid filler, diluent, encapsulating material orformulation auxiliary of any type. One exemplary pharmaceuticallyacceptable carrier is physiological saline. Other physiologicallyacceptable carriers and their formulations are known to one skilled inthe art and described, for example, in Remington's PharmaceuticalSciences, (18^(th) edition), A. Gennaro, 1990, Mack Publishing Company,Easton, Pa. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,sugars such as lactose, glucose and sucrose; starches such as cornstarch and potato starch; cellulose and its derivatives such as sodiumcarboxymethyl cellulose, ethyl cellulose and cellulose acetate; powderedtragacanth; malt; gelatin; talc; excipients such as cocoa butter andsuppository waxes; oils such as peanut oil, cottonseed oil; saffloweroil; sesame oil; olive oil; corn oil and soybean oil; glycols such aspropylene glycol; esters such as ethyl oleate and ethyl laurate; agar;detergents such as TWEEN™ 80; buffering agents such as magnesiumhydroxide and aluminum hydroxide; alginic acid; pyrogen-free water;isotonic saline; Ringer's solution; ethyl alcohol; and phosphate buffersolutions, as well as other non-toxic compatible lubricants such assodium lauryl sulfate and magnesium stearate, as well as coloringagents, releasing agents, coating agents, sweetening, flavoring andperfuming agents, preservatives and antioxidants can also be present inthe composition, according to the judgment of the formulator.

By “pharmaceutically acceptable salt” is meant non-toxic acid additionsalts or metal complexes which are commonly used in the pharmaceuticalindustry. Examples of acid addition salts include organic acids such asacetic, lactic, pamoic, maleic, citric, malic, ascorbic, succinic,benzoic, palmitic, suberic, salicylic, tartaric, methanesulfonic,toluenesulfonic, or trifluoroacetic acids or the like; polymeric acidssuch as tannic acid, carboxymethyl cellulose, or the like; and inorganicacids such as hydrochloric acid, hydrobromic acid, sulfuric acidphosphoric acid, or the like. Metal complexes include zinc, iron, andthe like.

The pharmaceutical compositions can be administered to a patient by anymeans known in the art including oral and parenteral routes. The term“patient”, as used herein, refers to humans as well as non-humans,including, for example, mammals, birds, reptiles, amphibians and fish.Preferably, the non-humans are mammals (e.g., a rodent (including amouse or rat), a rabbit, a monkey, a dog, a cat, sheep, cow, pig,horse). The non-human animal could alternatively be a bird, e.g., achicken or turkey.

In certain embodiments parenteral routes are preferred since they avoidcontact with the digestive enzymes that are found in the alimentarycanal. According to such embodiments, inventive compositions including atherapeutic agent may be administered by injection (e.g., intravenous,subcutaneous or intramuscular, intraperitoneal injection), rectally,vaginally, topically (as by powders, creams, ointments, or drops), or byinhalation (as by sprays), intranasal, pulmonary, or intrabuccal.

Injectable preparations, for example, sterile injectable aqueous oroleaginous suspensions, may be formulated according to the known artusing suitable dispersing or wetting agents and suspending agents. Thesterile injectable preparation may also be a sterile injectablesolution, suspension, or emulsion in a nontoxic parenterally acceptablediluent or solvent, for example, as a solution in 1,3-butanediol. Amongthe acceptable vehicles and solvents that may be employed are water,Ringer's solution, U.S.P. and isotonic sodium chloride solution. Inaddition, sterile, fixed oils are conventionally employed as a solventor suspending medium. For this purpose any bland fixed oil can beemployed including synthetic mono- or diglycerides. In addition, fattyacids such as oleic acid are used in the preparation of injectables. Ina particularly preferred embodiment, a therapeutic agent is suspended ina carrier fluid comprising 1% (w/v) sodium carboxymethyl cellulose and0.1% (v/v) TWEEN80™. The injectable formulations can be sterilized, forexample, by filtration through a bacteria-retaining filter, or byincorporating sterilizing agents in the form of sterile solidcompositions which can be dissolved or dispersed in sterile water orother sterile injectable medium prior to use.

Compositions for rectal or vaginal administration are preferablysuppositories which can be prepared by mixing the therapeutic agent withsuitable non-irritating excipients or carriers such as cocoa butter,polyethylene glycol, or a suppository wax which are solid at ambienttemperature but liquid at body temperature and therefore melt in therectum or vaginal cavity and release the therapeutic agent.

Dosage forms for topical or transdermal administration of apharmaceutical composition including a therapeutic agent includeointments, pastes, creams, lotions, gels, powders, solutions, sprays,inhalants, or patches. The therapeutic agent is admixed under sterileconditions with a pharmaceutically acceptable carrier and any neededpreservatives or buffers as may be required. Ophthalmic formulations,ear drops and eye drops are also contemplated as being within the scopeof this invention. The ointments, pastes, creams and gels may contain,in addition to the therapeutic agents of this invention, excipients suchas animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.Transdermal patches have the added advantage of providing controlleddelivery of a compound to the body. Such dosage forms can be made bydissolving or dispensing the therapeutic agents in a proper medium.Absorption enhancers can also be used to increase the flux of thecompound across the skin. The rate can be controlled by either providinga rate controlling membrane or by dispersing the therapeutic agents in apolymer matrix or gel.

Powders and sprays can also contain excipients such as lactose, talc,silicic acid, aluminum hydroxide, calcium silicates and polyamidepowder, or mixtures of these drugs. Sprays can additionally containcustomary propellants such as chlorofluorohydrocarbons.

When administered orally, the therapeutic agent is optionallyencapsulated. A variety of suitable encapsulation systems are known inthe art (“Microcapsules and Nanoparticles in Medicine and Pharmacy,”Edited by Doubrow, M., CRC Press, Boca Raton, 1992; Mathiowitz andLanger J. Control. Release 5:13, 1987; Mathiowitz et al., ReactivePolymers 6:275, 1987; Mathiowitz et al., J. Appl. Polymer Sci. 35:755,1988; Langer Acc. Chem. Res. 33:94, 2000; Langer J. Control. Release62:7, 1999; Uhrich et al., Chem. Rev. 99:3181, 1999; Zhou et al., J.Control. Release 75:27, 2001; and Hanes et al., Pharm. Biotechnol.6:389, 1995). For example, the therapeutic agent can be encapsulatedwithin biodegradable polymeric microspheres or liposomes. Examples ofnatural and synthetic polymers useful in the preparation ofbiodegradable microspheres include carbohydrates such as alginate,cellulose, polyhydroxyalkanoates, polyamides, polyphosphazenes,polypropylfumarates, polyethers, polyacetals, polycyanoacrylates,biodegradable polyurethanes, polycarbonates, polyanhydrides,polyhydroxyacids, poly(ortho esters) and other biodegradable polyesters.Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides and gangliosides.

Pharmaceutical compositions for oral administration can be liquid orsolid. Liquid dosage forms suitable for oral administration of inventivecompositions include pharmaceutically acceptable emulsions,microemulsions, solutions, suspensions, syrups and elixirs. In additionto an encapsulated or unencapsulated therapeutic agent, the liquiddosage forms may contain inert diluents commonly used in the art suchas, for example, water or other solvents, solubilizing agents andemulsifiers such as ethyl alcohol, isopropyl alcohol, ethyl carbonate,ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol,1,3-butylene glycol, dimethylformamide, oils (in particular, cottonseed,groundnut, corn, germ, olive, castor and sesame oils), glycerol,tetrahydrofurfuryl alcohol, polyethylene glycols and fatty acid estersof sorbitan and mixtures thereof. Besides inert diluents, the oralcompositions can also include adjuvants, wetting agents, emulsifying andsuspending agents, sweetening, flavoring and perfuming agents. As usedherein, the term “adjuvant” refers to any compound which is anonspecific modulator of the immune response. In certain preferredembodiments, the adjuvant stimulates the immune response. Any adjuvantmay be used in accordance with the present invention. A large number ofadjuvant compounds are known in the art (Allison, Dev. Biol. Stand.92:3, 1998; Unkeless et al., Annu. Rev. Immunol. 6:251, 1998; andPhillips et al., Vaccine 10: 151, 1992).

Solid dosage forms for oral administration include capsules, tablets,pills, powders and granules. In such solid dosage forms, theencapsulated or unencapsulated therapeutic agent is mixed with at leastone inert, pharmaceutically acceptable excipient or carrier such assodium citrate or dicalcium phosphate and/or (a) fillers or extenderssuch as starches, lactose, sucrose, glucose, mannitol and silicic acid,(b) binders such as, for example, carboxymethylcellulose, alginates,gelatin, polyvinylpyrrolidinone, sucrose and acacia, (c) humectants suchas glycerol, (d) disintegrating agents such as agar-agar, calciumcarbonate, potato or tapioca starch, alginic acid, certain silicates andsodium carbonate, (e) solution retarding agents such as paraffin, (f)absorption accelerators such as quaternary ammonium compounds, (g)wetting agents such as, for example, cetyl alcohol and glycerolmonostearate, (h) absorbents such as kaolin and bentonite clay and (i)lubricants such as talc, calcium stearate, magnesium stearate, solidpolyethylene glycols, sodium lauryl sulfate and mixtures thereof. In thecase of capsules, tablets and pills, the dosage form may also comprisebuffering agents.

Solid compositions of a similar type may also be employed as fillers insoft and hard-filled gelatin capsules using such excipients as lactoseor milk sugar as well as high molecular weight polyethylene glycols andthe like. The solid dosage forms of tablets, dragees, capsules, pillsand granules can be prepared with coatings and shells such as entericcoatings and other coatings well known in the pharmaceutical formulatingart.

The exact dosage of the therapeutic agent is chosen by the individualphysician in view of the patient to be treated. In general, dosage andadministration are adjusted to provide an effective amount of thetherapeutic agent to the patient being treated. As used herein, the“effective amount” of an therapeutic agent refers to the amountnecessary to elicit the desired biological response. As will beappreciated by those of ordinary skill in this art, the effective amountof therapeutic agent may vary depending on such factors as the desiredbiological endpoint, the drug to be delivered, the target tissue, theroute of administration, etc. For example, the effective amount oftherapeutic agent containing an anti-cancer drug might be the amountthat results in a reduction in tumor size by a desired amount over adesired period of time. Additional factors which may be taken intoaccount include the severity of the disease state; age, weight andgender of the patient being treated; diet, time and frequency ofadministration; drug combinations; reaction sensitivities; andtolerance/response to therapy. Long acting pharmaceutical compositionsmight be administered every 3 to 4 days, every week, or once every twoweeks depending on half-life and clearance rate of the particularcomposition.

The therapeutic agents of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of therapeutic agent appropriate for the patient to betreated. It will be understood, however, that the total daily usage ofthe compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. For anytherapeutic agent, the therapeutically effective dose can be estimatedinitially either in cell culture assays or in animal models, usuallymice, rabbits, dogs, or pigs. The animal model is also used to achieve adesirable concentration range and route of administration. Suchinformation can then be used to determine useful doses and routes foradministration in humans. Therapeutic efficacy and toxicity oftherapeutic agents can be determined by standard pharmaceuticalprocedures in cell cultures or experimental animals, e.g., ED50 (thedose is therapeutically effective in 50% of the population) and LD50(the dose is lethal to 50% of the population). The dose ratio of toxicto therapeutic effects is the therapeutic index and it can be expressedas the ratio, LD50/ED50. Pharmaceutical compositions which exhibit largetherapeutic indices are preferred. The data obtained from cell cultureassays and animal studies is used in formulating a range of dosage forhuman use.

If several different therapeutic modalities (e.g., with differenttherapeutic agents) are to be administered simultaneously then they maybe combined into a single pharmaceutical composition. Alternatively,they may be prepared as separate compositions that are then mixed orsimply administered one after the other. If several differenttherapeutic agents (e.g., with different therapeutic agents) are to beadministered at different times then they are preferably prepared asseparate compositions. If additional drugs are going to be included in acombination therapy they can be added to one or more of thesetherapeutic agents or prepared as separate compositions.

A peptide could be chemically modified in order to alter its propertiessuch as biodistribution, pharmacokinetics and solubility. Variousmethods have been used to increase the solubility and stability ofdrugs, among them the use of organic solvents, their incorporationwithin emulsions or liposomes, the adjustment of pH, their chemicalmodifications and their complexation with the cyclodextrins. Thecyclodextrins are oligosacharides cyclic family, which include six,seven or eight units of glucopyranose. Due to sterics interactions, thecyclodextrins form a cycle structure in the shape of a cone with aninternal cavity. Those are compounds chemically stable that can bemodified. The cyclodextrins hosts form complexes with varioushydrophobic guests in their cavity. The cyclodextrins are used for thesolubilization and encapsulation of drugs.

Liposomes and Controlled Release:

In order to design a drug delivery system, various kinds of highperformance carrier materials are being developed to deliver thenecessary amount of drug to the targeted site for a necessary period oftime, both efficiently and precisely.

Cyclodextrins, biodegradable or non biodegradable polymers, liposomes,emulsions. Multiple emulsions are potential candidates for such a role,because of their ability to alter physical, chemical and biologicalproperties of guest molecules.There are number of drug delivery systems including but not limited topolymer microcapsules, microparticles, nanoparticles, liposomes andemulsion. Many of these are prepared from synthetic biodegradablepolymers such as polyanhydrides and poly hydroxy acids. In these systemsthe drugs incorporate in polymeric microspheres, which release the druginside the organism in small and controlled daily doses during daysmonths or until years.

Several polymers already were tested in controlled release systems. Suchas: polyuretans for its elasticity, polysiloxans or silicons for being agood one insulating, polymethyl-metacrilate for its physical form;polyvinilalcohol for its hydrofobicity and resistance, polyethilene forits hardness and impermeability (Gilding, D. K. Biodegradable polymers.Biocompat. Clin. Impl. Mater. 2:209-232, 1981). Biodegradable polymersand biocompatible polymers, have been extensively investigated asvehicle for controlled release systems due to their ability to undergosurface degradation. These kind of polymers can be chose from:poly(2-hidroxi-ethylmetacrilate), polyacrilamide, polymer from lacticacid (PLA), from glicolic acid (PGA), and the respective onesco-polymers, (PLGA) and the poly(anidrides), as described by Tamada andLanger, J. Biomater. Sci. Polym. Edn, 3(4):315-353.

Suitable controlled release vehicles include, but are not limited to,biocompatible polymers, other polymeric matrices, capsules,microcapsules, nanocapsules, microparticles, nanoparticles, boluspreparations, osmotic pumps, diffusion devices, liposomes, lipospheresand transdermal delivery systems, implantable or not.

Satisfactory systems of controlled release include, but are not limitedto, the ciclodextrines, biocompatible polymers, biodegradable polymers,other polymeric matrixes, capsules, micro-capsules, microparticles,bolus preparations, osmotic pumps, diffusion devices, lipossomes,lipoesferes, and systems of transdermic administration. Othercompositions of controlled release include liquids that, when submittedthe temperature changes, form a solid or a gel in situ.

Liposomes are lipid vesicles that include aqueous internal compartmentsin which molecules, for example drugs, are encapsulated with theobjective of reaching a controlled release of the drug afteradministration in individuals. Many different techniques have beenproposed for the preparation of liposomes [U.S. Pat. No. 4,552,803,Lenk; U.S. Pat. No. 4,310,506, Baldeschwieler; U.S. Pat. No. 4,235,871,Papahadjopoulos; U.S. Pat. No. 4,224,179, Schneider; U.S. Pat. No.4,078,052, Papahadjopoulos; U.S. Pat. No. 4,394,372, Tailor; U.S. Pat.No. 4,308,166, Marchetti; U.S. Pat. No. 4,485,054, Mezei; and U.S. Pat.No. 4,508,703, Redziniak; Woodle and Papahadjopoulos, Methods Enzymol.171:193-215 (1989]; Unilamellar vesicles display a single membrane[Huang, Biochemistry 8:334-352 (1969] while muitilamellar vesicles(MLVs) have numerous concentric membranes [Bangham et al., J. Mol. Biol.13:238-252 (1965]. The procedure of Bangham [J. Mol. Biol. 13:238-252(1965] produces “ordinary MLVs”, that present unequal solutedistributions among the aqueous compartments and, consequently,differences of osmotic pressure. Lenk et al. (U.S. Pat. No. 4,522,803;U.S. Pat. No. 5,030,453 and U.S. Pat. No. 5,169,637), Fountain et al.(U.S. Pat. No. 4,588,578), Cullis et al. (U.S. Pat. No. 4,975,282) andGregoriadis et al. (Pat. W.O. 99/65465) introduced methods for thepreparation of MLVs that present substantially equal solutedistributions among the compartments. Similar solute distributions amongthe different compartments mean a larger drug encapsulation efficiencyas well as smaller differences of osmotic pressure that turns these MLVsmore stable than ordinary MLVs. Unilamellar vesicles can be produced bysonication of MLVs [Papahadjopoulos et al. (1968)] or by extrusionthrough polycarbonate membranes [Cullis et al. (U.S. Pat. No. 5,008,050)and Loughrey et al. (U.S. Pat. No. 5,059,421)].

Satisfactory lipids include for example, phosphatidylcholine,phosphatidylserine, phosphatidylglycerol, cardiolipin, cholesterol,phosphatidic acid, sphingolipids, glycolipids, fatty acids, sterols,phosphatidylethanolamine, polymerizable lipids in their polymerized ornon-polymerized form, mixture of these lipids.

The composition of the liposomes can be manipulated such as to turn themspecific for an organ or a cell type. The targeting of liposomes hasbeen classified either on the basis of anatomical factors or on thebasis of the mechanism of their interaction with the environment. Theanatomical classification is based on their level of selectivity, forexample, organ-specific or cell-specific. From the point of view of themechanisms, the—targeting can be considered as passive or active.

The passive targeting exploits the natural tendency of conventionalliposomes to be captured by the cells of the reticulo-endotheliaisystem, i.e. mainly the fixed macrophages in the liver, spleen and bonemarrow.

Sterically stabilized liposomes (also well-known as “PEG-liposomes”) arecharacterized by a reduced rate of elimination from the bloodcirculation [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., BocaRaton, Fla. (1995)].

PEG-liposomes present a polyethylene glycol polymer conjugated to thehead group of some phospholipid that reduces their interaction withplasma proteins, such as opsonins, and reduces the rate of their uptakeby cells. The resulting steric barrier allows these liposomes to remainfor a longer period of time within the circulation than conventionalliposomes [Lasic and Martin, Stealth Liposomes, CRC Press, Inc., BocaRaton, Fla. (1995); Woodle et al., Biochim. Biophys. Acta 1105:193-200(1992); Litzinger et al., Biochim. Biophys. Acta 1190:99-107 (1994);Bedu Addo, et al., Pharm. Res. 13:718-724 (1996]. The drug encapsulationwithin PEG-liposomes has resulted in the improvement of theeffectiveness of many chemotherapeutic agents [Lasic and Martin, Stealthliposomes, CRC Press, Inc., Boca Raton, Fla. (1995)] and bioactivepeptides [Allen T. M. In: Liposomes, New Systems, New Trends in theirApplications (F. Puisieux, P. Couvreur, J. Delattre, J.-P. DevissaguetEd.), Editions de la Sante, France, 1995, pp. 125].

Studies in this area demonstrated that different factors affect theeffectiveness of PEG-liposomes. Ideally, the diameter of the vesiclesshould be below 200 nm, the number of units in PEG of approximately2.000 and the proportion of Pegylated lipid from 3 to 5 mol % [Lasic andMartin, Stealth Liposomes, CRC Press, Inc., Boca Raton, Fla. (1995);Woodle et al., Biochim. Biophys. Acta 1105:193-200 (1992); Litzinger etal., Biochim. Biophys. Acta 1190:99-107(1994); Bedu Addo et al., Pharm.Res. 13:718-724(1996)].

The active targeting involves alteration of liposomes through theirassociation with a liguand, such as a monoclonal antibody, a sugar, aglycolipid, protein, a polymer or by changing the lipid composition orthe liposome size to target them to organs and cells different fromthose which accumulate conventional liposomes.

Mucosal Delivery Enhancing Agents

“Mucosal delivery enhancing agents” are defined as chemicals and otherexcipients that, when added to a formulation comprising water, saltsand/or common buffers and peptide within the present invention (thecontrol formulation) produce a formulation that produces a significantincrease in transport of peptide across a mucosa as measured by themaximum blood, serum, or cerebral spinal fluid concentration (Cmax) orby the area under the curve, AUC, in a plot of concentration versustime. A mucosa includes the nasal, oral, intestional, buccal,bronchopulmonary, vaginal, and rectal mucosal surfaces and in factincludes all mucus-secreting membranes lining all body cavities orpassages that communicate with the exterior. Mucosal delivery enhancingagents are sometimes called carriers.

Compositions and Methods of Sustained Release

The present invention provides improved mucosal (e.g., nasal) deliveryof a formulation comprising the peptide within the present invention incombination with one or more mucosal delivery-enhancing agents and anoptional sustained release-enhancing agent or agents. Mucosaldelivery-enhancing agents of the present invention yield an effectiveincrease in delivery, e.g., an increase in the maximal plasmaconcentration (Cmax) to enhance the therapeutic activity ofmucosally-administered peptide. A second factor affecting therapeuticactivity of the peptide in the blood plasma and CNS is residence time(RT). Sustained release-enhancing agents, in combination with intranasaldelivery-enhancing agents, increase Cmax and increase residence time(RT) of the peptide. Polymeric delivery vehicles and other agents andmethods of the present invention that yield sustained release-enhancingformulations, for example, polyethylene glycol (PEG), are disclosedherein. Within the mucosal delivery formulations and methods of theinvention, the peptide is frequently combined or coordinatelyadministered with a suitable carrier or vehicle for mucosal delivery. Asused herein, the term “carrier” means a pharmaceutically acceptablesolid or liquid filler, diluent or encapsulating material. Awater-containing liquid carrier can contain pharmaceutically acceptableadditives such as acidifying agents, alkalizing agents, antimicrobialpreservatives, antioxidants, buffering agents, chelating agents,complexing agents, solubilizing agents, humectants, solvents, suspendingand/or viscosity-increasing agents, tonicity agents, wetting agents orother biocompatible materials. A tabulation of ingredients listed by theabove categories, can be found in the U.S. Pharmacopeia NationalFormulary, 1857-1859, (1990). As used herein, “mucosaldelivery-enhancing agents” include agents which enhance the release orsolubility (e.g., from a formulation delivery vehicle), diffusion rate,penetration capacity and timing, uptake, residence time, stability,effective half-life, peak or sustained concentration levels, clearanceand other desired mucosal delivery characteristics (e.g., as measured atthe site of delivery, or at a selected target site of activity such asthe bloodstream or central nervous system) of the peptide or otherbiologically active compound(s). Within certain aspects of theinvention, absorption-promoting agents for coordinate administration orcombinatorial formulation with the peptide of the invention are selectedfrom small hydrophilic molecules, including but not limited to, dimethylsulfoxide (DMSO), dimethylformamide, ethanol, propylene glycol, and the2-pyrrolidones. Alternatively, long-chain amphipathic molecules, forexample, deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleicacid, and the bile salts, may be employed to enhance mucosal penetrationof the peptide. In additional aspects, surfactants (e.g., polysorbates)are employed as adjunct compounds, processing agents, or formulationadditives to enhance intranasal delivery of the peptide. Agents such asDMSO, polyethylene glycol, and ethanol can, if present in sufficientlyhigh concentrations in delivery environment (e.g., by pre-administrationor incorporation in a therapeutic formulation), enter the aqueous phaseof the mucosa and alter its solubilizing properties, thereby enhancingthe partitioning of the peptide from the vehicle into the mucosa. Themucosal therapeutic and prophylactic compositions of the presentinvention may be supplemented with any suitable penetration-promotingagent that facilitates absorption, diffusion, or penetration of thepeptide across mucosal barriers. The penetration promoter may be anypromoter that is pharmaceutically acceptable.

Charge Modifying and pH Control Agents and Methods

To improve the transport characteristics of biologically active agents(including the peptide within the present invention), for enhanceddelivery across hydrophobic mucosal membrane barriers, the inventionalso provides techniques and reagents for charge modification ofselected biologically active agents or delivery-enhancing agentsdescribed herein. In this regard, the relative permeabilities ofmacromolecules is generally be related to their partition coefficients.The degree of ionization of molecules, which is dependent on the pKa ofthe molecule and the pH at the mucosal membrane surface, also affectspermeability of the molecules. Permeation and partitioning ofbiologically active agents, including the peptide within the presentinvention, for mucosal delivery may be facilitated by charge alterationor charge spreading of the active agent or permeabilizing agent, whichis achieved, for example, by alteration of charged functional groups, bymodifying the pH of the delivery vehicle or solution in which the activeagent is delivered, or by coordinate administration of a charge- orpH-altering reagent with the active agent. Consistent with these generalteachings, mucosal delivery of charged macromolecular species, includingthe peptide within the present invention is substantially improved whenthe active agent is delivered to the mucosal surface in a substantiallyun-ionized, or neutral, electrical charge state.

Certain peptide and protein components of mucosal formulations for usewithin the invention will be charge modified to yield an increase in thepositive charge density of the peptide or protein. These modificationsextend also to cationization of peptide and protein conjugates, carriersand other delivery forms disclosed herein.

Degradative Enzyme Inhibitory Agents and Methods

Another excipient that may be included in a trans-mucosal preparation isa degradative enzyme inhibitor. Any inhibitor that inhibits the activityof an enzyme to protect the biologically active agent(s) may be usefullyemployed in the compositions and methods of the invention. Useful enzymeinhibitors for the protection of biologically active proteins andpeptides include, for example, soybean trypsin inhibitor, pancreatictrypsin inhibitor, chymotrypsin inhibitor and trypsin and chrymotrypsininhibitor isolated from potato (solanum tuberosum L.) tubers. Acombination or mixtures of inhibitors may be employed. The inhibitor(s)may be incorporated in or bound to a carrier, e.g., a hydrophilicpolymer, coated on the surface of the dosage form which is to contactthe nasal mucosa, or incorporated in the superficial phase of thesurface, in combination with the biologically active agent or in aseparately administered (e.g., pre-administered) formulation. Additionalenzyme inhibitors for use within the invention are selected from a widerange of non-protein inhibitors that vary in their degree of potency andtoxicity. As described in further detail below, immobilization of theseadjunct agents to matrices or other delivery vehicles, or development ofchemically modified analogues, may be readily implemented to reduce oreven eliminate toxic effects, when they are encountered. Among thisbroad group of candidate enzyme inhibitors for use within the inventionare organophosphorous inhibitors, such as diisopropylfluorophosphate(DFP) and phenylmethylsulfonyl fluoride (PMSF), which are potent,irreversible inhibitors of serine proteases (e.g., trypsin andchymotrypsin). Yet another type of enzyme inhibitory agent for usewithin the methods and compositions of the invention are amino acids andmodified amino acids that interfere with enzymatic degradation ofspecific therapeutic compounds.

The therapeutic agents of the invention can be used to treat disordersfor which modulation of GPCR-related signal transduction pathways isefficacious. For example, the peptides of the invention falling withinFormula I are used to treat disorders for which modulation or activationof relaxin-related GPCR receptors and/or LGR family of GPCRs responsesin a cell or in a subject is efficacious. Examples of such peptides aredepicted in SEQ ID NOs: 1-15, 20-26. In another example, the peptide ofthe invention falling within Formula I are further used to treat anydisease or condition that involves agonist associated activation and/ormodulation of relaxin or relaxin related peptides activity onrelaxin-related family of GPCRs and/or LGR family of GPCRs, either byitself or in combination with relaxin or any relaxin related peptide oranother compound, wherein the activation and/or modulation of thereceptor can be either by direct activation of downstream pathwaysdirectly related to the receptors or to G-proteins activated by thereceptors or any other related pathway, or by indirect activation byeither relaxin or any relaxin related peptide or another compound. Thedisorder is selected from but not limited to hyperplastic disorders,neoplastic disorders, cancer; fibrotic conditions, disorders of collagendeposition, fibrotic breakdown, connective tissue remodeling,uncontrolled or abnormal collagen or fibronectin formation or breakdown;skin injuries including wound healing and scarring, scleroderma;urogenital disorders including female reproductive disorders, male andfemale infertility, cryptorchidism, disregulation of spermatogenesis andreproductive development including descent of the gonads; conditionsassociated with pregnancy such as preeclampsia or complication of labor;angiogenesis related disorders; cardiovascular disorders,vasodilatation, vasoconstriction or hypertension, endothelialdisfunction and vascular disease, congestive heart failure, coronaryartery disease, ischemia and ischemia-reperfusion, peripheral vasculardisease; kidney disease, renal disease associated with arteriosclerosisor other narrowing of kidney capillaries; capillaries narrowing in thebody, such as in the eyes or in the peripheral digits, the mesocaecum,lung and peripheral vasculature; CNS related disorders, neurologicaldisorders, cognition and memory related indications, depression,neurological modification; inflammatory disorders, such as gastritis,gout, gouty arthritis, arthritis, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, ulcers; autoimmunedisorders; inflammatory conditions associated with viral infection andinfection related diseases including fibrosis and cirrhosis; Raynaud'sdisease, Raynaud's phenomenon; bone related conditions includingosteoporosis; metabolic disorders including food and water intake,diabetes, obesity; respiratory or a pulmonary disorder, includingasthma, COPD, bronchial disease, lung diseases, cystic fibrosis, ARDS,SARS.

In another aspect, the invention provides a method of treating,preventing or ameliorating the symptoms of a relaxin-related GPCR familyor LGR family of GPCRs-mediated or related disorders or conditions in apatient, the disorder or condition selected from the group consisting ofbut not limited to hyperplastic disorders, neoplastic disorders, cancer;fibrotic conditions, disorders of collagen deposition, fibroticbreakdown, connective tissue remodeling, uncontrolled or abnormalcollagen or fibronectin formation or breakdown; skin injuries includingwound healing and scarring, scleroderma; urogenital disorders includingfemale reproductive disorders, male and female infertility,cryptorchidism, disregulation of spermatogenesis and reproductivedevelopment including descent of the gonads; conditions associated withpregnancy such as preeclampsia or complication of labor; angiogenesisrelated disorders; cardiovascular disorders, vasodilatation,vasoconstriction or hypertension, endothelial disfunction and vasculardisease, congestive heart failure, coronary artery disease, ischemia andischemia-reperfusion, peripheral vascular disease; kidney disease, renaldisease associated with arteriosclerosis or other narrowing of kidneycapillaries; capillaries narrowing in the body, such as in the eyes orin the peripheral digits, the mesocaecum, lung and peripheralvasculature; CNS related disorders, neurological disorders, cognitionand memory related indications, depression, neurological modification;inflammatory disorders, such as gastritis, gout, gouty arthritis,arthritis, rheumatoid arthritis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, ulcers; autoimmune disorders; inflammatoryconditions associated with viral infection and infection relateddiseases including fibrosis and cirrhosis; Raynaud's disease, Raynaud'sphenomenon; bone related conditions including osteoporosis; metabolicdisorders including food and water intake, diabetes, obesity;respiratory or a pulmonary disorder, including asthma, COPD, bronchialdisease, lung diseases, cystic fibrosis, ARDS, SARS,

the method comprising administering to the patient an effective amountof a peptide of the invention.

In a further aspect, the invention contemplates the use of the analoguesand/or pharmaceutical compositions of the present invention in themanufacture of a medicament for the treatment of any disease orcondition that involves relaxin-related family of GPCRs or LGR GPCRsfamily related disorder or condition in a patient, the disorder selectedfrom the group consisting of but not limited to: hyperplastic disorders,neoplastic disorders, cancer; fibrotic conditions, disorders of collagendeposition, fibrotic breakdown, connective tissue remodeling,uncontrolled or abnormal collagen or fibronectin formation or breakdown;skin injuries including wound healing and scarring, scleroderma;urogenital disorders including female reproductive disorders, male andfemale infertility, cryptorchidism, disregulation of spermatogenesis andreproductive development including descent of the gonads; conditionsassociated with pregnancy such as preeclampsia or complication of labor;angiogenesis related disorders; cardiovascular disorders,vasodilatation, vasoconstriction or hypertension, endothelialdisfunction and vascular disease, congestive heart failure, coronaryartery disease, ischemia and ischemia-reperfusion, peripheral vasculardisease; kidney disease, renal disease associated with arteriosclerosisor other narrowing of kidney capillaries; capillaries narrowing in thebody, such as in the eyes or in the peripheral digits, the mesocaecum,lung and peripheral vasculature; CNS related disorders, neurologicaldisorders, cognition and memory related indications, depression,neurological modification; inflammatory disorders, such as gastritis,gout, gouty arthritis, arthritis, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, ulcers; autoimmunedisorders; inflammatory conditions associated with viral infection andinfection related diseases including fibrosis and cirrhosis; Raynaud'sdisease, Raynaud's phenomenon; bone related conditions includingosteoporosis; metabolic disorders including food and water intake,diabetes, obesity; respiratory or a pulmonary disorder, includingasthma, COPD, bronchial disease, lung diseases, cystic fibrosis, ARDS,SARS.

The term “relaxin-related family of GPCRs or LGR GPCRs family relateddisorder or condition” as used herein refers to conditions whererelaxin-related family of GPCRs or LGR GPCRs family are involved orimplicated in the development, maintenance or progression of the diseaseor condition.

The peptides of the invention falling within Formula I such as forexample, peptides as depicted in SEQ ID NOs:1-15, 20-26 are also usefulfor the treatment of fibrotic conditions involving tissue remodelingfollowing inflammation or ischemia-reperfusion injury, including but notlimited to endomyocardial and cardiac fibrosis fibrosis; mediastinalfibrosis; idiopathy pulmonary fibrosis; pulmonary fibrosis;retroperitoneal fibrosis; fibrosis of the spleen; fibrosis of thepancreas; hepatic fibrosis (cirrhosis) alcohol and non-alcohol related(including viral infection such as HAV, HBV and HCV); fibromatosis;granulomatous lung disease; glomerulonephritis, myocardial scarringfollowing infarction; endometrial fibrosis and endometriosis; woundhealing whether by injury or surgical procedures, diabetes related woundfibrosis.

The peptides of the invention falling within Formula I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26 are also usefulin treating cardiovascular diseases and their-complications, peripheralvascular diseases and coronary artery diseases, including but notlimited to myocardial infarction; congestive heart failure (CHF);myocardial failure; myocardial hypertrophy; ischemic cardiomyopathy;systolic heart failure; diastolic heart failure; stroke; thromboticstroke; concentric LV hypertrophy, myocarditis; cardiomyopathy;hypertrophic cardiomyopathy; myocarditis; decompensated heart failure;ischemic myocardial disease; congenital heart disease; angina pectoris;prevention of heart remodeling or ventricular remodeling aftermyocardial infarction; ischemia-reperfusion injury in ischemic andpost-ischemic events (e.g. myocardial infarct); cerebrovascularaccident; mitral valve regurgitation; hypertension; hypotension;restenosis; fibrosis; thrombosis; or platelet aggregation.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26 are useful intreating ischemia-reperfusion injury associated with ischemic andpost-ischemic events in organs and tissues, including but not limited tothrombotic stroke; myocardial infarction; angina pectoris; embolicvascular occlusions; peripheral vascular insufficiency; splanchnicartery occlusion; arterial occlusion by thrombi or embolisms, arterialocclusion by non-occlusive processes such as following low mesentericflow or sepsis; mesenteric arterial occlusion; mesenteric veinocclusion; ischemia-reperfusion injury to the mesentericmicrocirculation; ischemic acute renal failure; ischemia-reperfusioninjury to the cerebral tissue; intestinal intussusception; hemodynamicshock; tissue dysfunction; organ failure; restenosis; atherosclerosis;thrombosis; platelet aggregation.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are useful intreating ischemia-reperfusion injury following conditions including butnot limited to procedures such as cardiac surgery; organ surgery; organtransplantation; angiography; cardiopulmonary and cerebralresuscitation.

In another aspect, the peptides of the invention falling within FormulasI, such as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26,are used for prevention and treatment of hypertension and itscomplications including but not limited to hypertensive heart disease;antihypertension (blood pressure reduction); systemic and pulmonary highblood pressure; cerebrovascular disease and stroke; heart failure andstroke; left ventricular hypertrophy (LVH); congestive heart failure(CHF); hypertension, high blood pressure; vasodilation; renalhypertension; diuresis; nephritis; natriuresis; scleroderma renalcrisis; angina pectoris (stable and unstable); myocardial infarction;heart attack; coronary artery disease; coronary heart disease; cardiacarrhythmias; atrial fibrillation; portal hypertension; raisedintraocular pressure; vascular restenosis; chronic hypertension;valvular disease; myocardial ischemia; acute pulmonary edema; acutecoronary syndrome; hypertensive retinopathy; hypertensive pregnancysickness; preeclampsia; Raynaud's phenomenon; erectile dysfunction andglaucoma. These peptides are also used as a vasodilator and inantithrombotic therapy.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are alsouseful in treating inflammatory conditions associated with an infection,e.g., a bacterial infection or a viral infection, including but notlimited to a viral infection caused by human immunodeficiency virus I(HIV-1) or HIV-2, acquired immune deficiency (AIDS), West Nileencephalitis virus, coronavirus, rhinovirus, influenza virus, denguevirus, HCV, HBV, HAV, hemorrhagic fever; an otological infection; severeacute respiratory syndrome (SARS), sepsis and sinusitis.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are alsouseful in the prevention or treatment of cancer, or inflammationassociated with cancer such as solid cancer, including but not limitedto colon cancer, lung cancer, breast cancer, prostate cancer, braincancer, pancreatic cancer, ovarian cancer, kidney cancer, testicularcancer, bone cancer, osteosarcoma, or liver cancer (HBV/HCV related ornon-related). The cancer can alternatively be a melanoma, glioma, asarcoma, a leukemia, or lymphoma. These peptides are also useful in theprevention or treatment of invasive and metastatic cancer.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are used forthe prevention and treatment in skin injuries, including but not limitedto dermal repair, wound healing; burns, erythemas, lesions, woundhealing following surgical procedures; skin or tissue lesions includinglesions induced by conditions including, but not limited to Psoriasis,Lupus and Kaposhi Sarcoma; Scleroderma and collagenous diseases of theskin and skin tumors.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are used inthe prevention or treatment of a urogenital disorder or agenitor-urological disorders including but not limited to renal disease;a bladder disorder; disorders of the reproductive system; gynecologicdisorders; urinary tract disorder; incontinence; disorders of the male(spermatogenesis, spermatic motility), and female reproductive system;sexual dysfunction; erectile dysfunction; embryogenesis; and conditionsassociated with pregnancy. These are also used in pregnancy monitoring.As used herein, the term “conditions associated with pregnancy”includes, but is not limited to, conditions of fertilisation, pregnancy,parturition and lactation. The invention in another embodiment includesusing the peptides of the invention falling within Formulas I, such asfor example, peptides as depicted in SEQ ID NOs: 1-15, 20-26, whereinsaid pregnancy related disorders are selected from a group consisting ofAbnormal endometrial angiogenesis; Placental development defects;Cervical ripening (softening); Abnormal implantation; Nipple developmentand disfunction; Pregnancy related remodeling of the Uterine tissue;Endometriosis; Preeclampsia; Lactation disorders; Estrogenic andnon-estrogenic related hormonal disorders; Pre-term labor; post termlabor; and Labor complications.

The invention also provides peptides falling within Formulas I, such asfor example, peptides as depicted in SEQ ID NOs: 1-15, 20-26, that areused in the prevention or treatment of respiratory diseases, includingbut not limited to asthma, bronchial disease, lung diseases, chronicobstructive pulmonary disease (COPD), Acute Respiratory DistressSyndrome (ARDS), severe acute respiratory syndrome (SARS), Fibrosisrelated Asthma, cystic fibrosis.

The invention also provides peptides falling within Formulas I, such asfor example, peptides as depicted in SEQ ID NOs: 1-15, 20-26, that areused in the prevention or treatment of metabolic disorders including butnot limited to diabetes, diabetes mellitus, lipodystrophy,hyperthyroidism, glaucoma, hyperlipidaemia, non-insulin dependentdiabetes, Food intake; Water intake; Feeding and drinking behaviors,Anorexia, Cachexia (cancer and non cancer related); Fat and lipidmetabolism; and Energy control, appetite control and obesity.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedin the prevention and treatment of kidney diseases including but notlimited to diabetic nephropathy; glomerulosclerosis; nephropathies;renal impairment; scleroderma renal crisis and chronic renal failure.These peptides can also be used as antidiuretics.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedin the prevention and treatment of angiogenesis related conditionsincluding but not limited to retinal angiogenesis in a number of humanocular diseases such as diabetes mellitus, retinopathy of prematury, andage-related macular degeneration, or cancer associated angiogenesis inprimary or metastatic cancer, including but not limited to cancer of theprostate, brain, breast, colorectal, lung, ovarian, pancreatic, renal,cervical, melanoma, soft tissue sarcomas, lymphomas, head-and-neck, andglioblastomas.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedto treat a central nervous system (CNS) disorder, including but notlimited to central and peripheral degenerative neuropathies;neuroprotection; impaired cognition; anxiety disorders, pain control,food intake, a behavioral disorder, a learning disorder, a sleepdisorder, a memory disorder, a pathologic response to anesthesia,addiction, depression, migraine, a menstruation disorder, muscle spasm,opiate dependence, dementia, Alzheimer's disease, Parkinson's disease,cortical function, locomotor activity, Alcohol and Drug addiction andabuse; Impared memory; Feeding and drinking related behaviours; Stresscontrol, Bipolar disorder; Schyzophrenia; Schyzoaffective; MultipleSclerosis (MS); Stroke and stroke damage repair (Ischemia protection);Vasculature and re-vasculature in the brain; and Brain tissueregeneration and a peripheral nervous system disorder.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedto treat a bone and bone related disorders in a patient, including butnot limited to Osteoporosis; Osteoarthritis; Osteopetrosis; Boneinconsistency; Osteosarcoma; and Cancer matastesis to the bone.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedto treat endothelial dysfunction disease, selected from a groupconsisting of cardiovascular diseases, high blood pressure,atherosclerosis, thrombosis, myocardial infarct, heart failure, renaldiseases, plurimetabolic syndrome, erectile dysfunction; vasculitis; anddiseases of the central nervous system (CNS).

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedto treat inflammatory disorder in a subject. The inflammatory disordercan be gastritis, gout, gouty arthritis, arthritis, rheumatoidarthritis, inflammatory bowel disease, Crohn's disease, ulcerativecolitis, ulcers, chronic bronchitis, asthma, allergy, acute lung injury,pulmonary inflammation, airway hyper-responsiveness, vasculitis, septicshock and inflammatory skin disorders, including but not limited topsoriasis, atopic dermatitis, eczema.

The peptides of the invention falling within Formulas I, such as forexample, peptides as depicted in SEQ ID NOs: 1-15, 20-26, are also usedto treat autoimmune disease or disorder in a subject. The autoimmunedisease can be multiple sclerosis, psoriasis, rheumatoid arthritis,systemic lupus erythematosus, ulcerative colitis, Crohn's disease,transplant rejection, immune disorders associated with grafttransplantation rejection, benign lymphocytic angiitis, lupuserythematosus, Hashimoto's thyroiditis, primary myxedema, Graves'disease, pernicious anemia, autoimmune atrophic gastritis, Addison'sdisease, insulin dependent diabetes mellitis, Good pasture's syndrome,myasthenia gravis, pemphigus, sympathetic ophthalmia, autoimmuneuveitis, autoimmune hemolytic anemia, idiopathic thrombocytopenia,primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis,Sjogren's syndrome, rheumatic disease, polymyositis, scleroderma, mixedconnective tissue disease, inflammatory rheumatism, degenerativerheumatism, extra-articular rheumatism, collagen diseases, chronicpolyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenilerheumatoid arthritis, periarthritis humeroscapularis, panarteriitisnodosa, progressive systemic scleroderma, arthritis uratica,dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

Compounds of Formula I can be used to treat disorders, diseases and/orconditions as described herein, by administering to a subject in needthereof a therapeutically effective amount of a peptide falling withinFormula I.

Also provided by the invention is a method of treating disorders forwhich modulation of GPCR-related signal transduction pathways isefficacious. For example, provided by the invention is a method oftreating disorders for which modulation of relaxin-related family ofGPCR and/or LGR family of GPCRs is efficacious in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formula I, such as for example,peptides as depicted in SEQ ID NOs: 1-15, 20-26. Such diseases disordersand/or condition are selected from but not limited to hyperplasticdisorders, neoplastic disorders, cancer; fibrotic conditions, disordersof collagen deposition, fibrotic breakdown, connective tissueremodeling, uncontrolled or abnormal collagen or fibronectin formationor breakdown; skin injuries including wound healing and scarring,scleroderma; urogenital disorders including female reproductivedisorders, male and female infertility, cryptorchidism, disregulation ofspermatogenesis and reproductive development including descent of thegonads; conditions associated with pregnancy such as preeclampsia orcomplication of labor; angiogenesis related disorders; cardiovasculardisorders, vasodilatation, vasoconstriction or hypertension, endothelialdisfunction and vascular disease, congestive heart failure, coronaryartery disease, ischemia and ischemia-reperfusion, peripheral vasculardisease; kidney disease, renal disease associated with arteriosclerosisor other narrowing of kidney capillaries; capillaries narrowing in thebody, such as in the eyes or in the peripheral digits, the mesocaecum,lung and peripheral vasculature; CNS related disorders, neurologicaldisorders, cognition and memory related indications, depression,neurological modification; inflammatory disorders, such as gastritis,gout, gouty arthritis, arthritis, rheumatoid arthritis, inflammatorybowel disease, Crohn's disease, ulcerative colitis, ulcers; autoimmunedisorders; inflammatory conditions associated with viral infection andinfection related diseases including fibrosis and cirrhosis; Raynaud'sdisease, Raynaud's phenomenon; bone related conditions includingosteoporosis; metabolic disorders including food and water intake,diabetes, obesity; respiratory or a pulmonary disorder, includingasthma, COPD, bronchial disease, lung diseases, cystic fibrosis, ARDS,SARS.

Also provided by the invention is a method of treating an inflammatorydisorder in a subject by administering to a subject in need thereof atherapeutically effective amount of a compound falling within Formula I,such as for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26.The inflammatory disorder can be gastritis, gout, gouty arthritis,arthritis, rheumatoid arthritis, inflammatory bowel disease, Crohn'sdisease, ulcerative colitis, ulcers, chronic bronchitis, asthma,allergy, acute lung injury, pulmonary inflammation, airwayhyper-responsiveness, vasculitis, septic shock and inflammatory skindisorders, including but not limited to psoriasis, atopic dermatitis,eczema.

Also provided by the invention is a method of treating fibroticconditions involving tissue remodeling in a subject by administering toa subject in need thereof a therapeutically effective amount of acompound falling within Formula I such as for example, peptides asdepicted in SEQ ID NOs: 1-15, 20-26. These fibrotic conditions can beendomyocardial and cardiac fibrosis fibrosis; mediastinal fibrosis;idiopathy pulmonary fibrosis; pulmonary fibrosis; retroperitonealfibrosis; fibrosis of the spleen; fibrosis of the pancreas; hepaticfibrosis (cirrhosis) alcohol and non-alcohol related (including viralinfection such as HAV, HBV and HCV); fibromatosis; granulomatous lungdisease; glomerulonephritis, myocardial scarring following infarction;endometrial fibrosis and endometriosis; wound healing whether by injuryor surgical procedures, diabetes related wound fibrosis.

Also provided by the invention is a method of treating an autoimmunedisease or disorder in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormula I, such as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26. The autoimmune disease can be multiple sclerosis,psoriasis, rheumatoid arthritis, systemic lupus erythematosus,ulcerative colitis, Crohn's disease, transplant rejection, immunedisorders associated with graft transplantation rejection, benignlymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis,primary myxedema, Graves' disease, pernicious anemia, autoimmuneatrophic gastritis, Addison's disease, insulin dependent diabetesmellitis, Good pasture's syndrome, myasthenia gravis, pemphigus,sympathetic ophthalmia, autoimmune uveitis, autoimmune hemolytic anemia,idiopathic thrombocytopenia, primary biliary cirrhosis, chronic actionhepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease,polymyositis, scleroderma, mixed connective tissue disease, inflammatoryrheumatism, degenerative rheumatism, extra-articular rheumatism,collagen diseases, chronic polyarthritis, psoriasis arthropathica,ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritishumeroscapularis, panarteriitis nodosa, progressive systemicscleroderma, arthritis uratica, dermatomyositis, muscular rheumatism,myositis, myogelosis and chondrocalcinosis.

Also provided by the invention is a method of treating cardiovasculardiseases and their complications in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formula I, such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The cardiovascular diseases can be peripheralvascular diseases and coronary artery diseases, including but notlimited to myocardial infarction; coronary heart disease; congestiveheart failure (CHF); myocardial failure; myocardial hypertrophy;ischemic cardiomyopathy; systolic heart failure; diastolic heartfailure; stroke; thrombotic stroke; concentric LV hypertrophy,myocarditis; cardiomyopathy; hypertrophic cardiomyopathy; myocarditis;decompensated heart failure; ischemic myocardial disease; congenitalheart disease; angina pectoris; prevention of heart remodeling orventricular remodeling after myocardial infarction; ischemia-reperfusioninjury in ischemic and post-ischemic events (e.g. myocardial infarct);cerebrovascular accident; mitral valve regurgitation; hypertension;hypotension; restenosis; fibrosis; thrombosis; or platelet aggregation.

Also provided by the invention is a method of treating anischemia-reperfusion injury in a subject by administering to a subjectin need thereof a therapeutically effective amount of a compound fallingwithin Formula I, such as for example, peptides as depicted in SEQ IDNOs: 1-15, 20-26. The ischemia-reperfusion injury can be associated withischemic and post-ischemic events in organs and tissues, including butnot limited to thrombotic stroke; myocardial infarction; anginapectoris; embolic vascular occlusions; peripheral vascularinsufficiency; splanchnic artery occlusion; arterial occlusion bythrombi or embolisms, arterial occlusion by non-occlusive processes suchas following low mesenteric flow or sepsis; mesenteric arterialocclusion; mesenteric vein occlusion; ischemia-reperfusion injury to themesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation. Theischemia-reperfusion injury can be alternatively following conditionsincluding but not limited to procedures such as cardiac surgery; organsurgery; organ transplantation; angiography; cardiopulmonary andcerebral resuscitation.

Also provided by the invention is a method of preventing and treating anhypertension and its complications in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formula I, such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The hypertension and its complications can behypertensive heart disease; antihypertension (blood pressure reduction);systemic and pulmonary high blood pressure; cerebrovascular disease andstroke; heart failure and stroke; left ventricular hypertrophy (LVH);congestive heart failure (CHF); hypertension, high blood pressure;vasodilation; renal hypertension; diuresis; nephritis; natriuresis;scleroderma renal crisis; angina pectoris (stable and unstable);myocardial infarction; heart attack; coronary artery disease; coronaryheart disease; cardiac arrhythmias; atrial fibrillation; portalhypertension; raised intraocular pressure; vascular restenosis; chronichypertension; valvular disease; myocardial ischemia; acute pulmonaryedema; acute coronary syndrome; hypertensive retinopathy; hypertensivepregnancy sickness; preeclampsia; Raynaud's phenomenon; erectiledysfunction and glaucoma. These peptides are also used as a vasodilatorand in antithrombotic therapy.

Also provided by the invention is a method of treating an inflammatorydisorder and/or conditions associated with an infection in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I, such as for example,peptides as depicted in SEQ ID NOs: 1-15, 20-26. The inflammatoryconditions associated with an infection, can be a bacterial infection ora viral infection, including but not limited to a viral infection causedby human immunodeficiency virus I (HIV-1) or HIV-2, acquired immunedeficiency (AIDS), HAV, HCV, HBV, West Nile encephalitis virus,coronavirus, rhinovirus, influenza virus, dengue virus, hemorrhagicfever; an otological infection; severe acute respiratory syndrome(SARS), sepsis and sinusitis.

Also provided by the invention is a method of treating cancer, orinflammation associated with cancer in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I, such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The cancer, or inflammation associated withcancer can be solid cancer, including but not limited to colon cancer,lung cancer, breast cancer, prostate cancer, brain cancer, pancreaticcancer, ovarian cancer, testicular cancer, bone cancer, osteosarcoma,liver cancer (HBV/HCV related or non-related), or kidney cancer. Thecancer can alternatively be a melanoma, glioma, a sarcoma, a leukemia,or lymphoma. These peptides are also useful in the prevention ortreatment of invasive and metastatic cancer.

Also provided by the invention is a method of treating skin injurydiseases, disorders and/or conditions in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I, such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The skin injury diseases, disorders and/orconditions can be dermal repair, wound healing; burns, erythemas,lesions, wound healing following surgical procedures; skin or tissuelesions including lesions induced by conditions including, but notlimited to Psoriasis, Lupus and Kaposhi Sarcoma; Scleroderma andcollagenous diseases of the skin and skin tumors.

Also provided by the invention is a method of prevention or treatment ofa urogenital disorder or a genitor-urological disorders in a subject byadministering to a subject in need thereof a therapeutically effectiveamount of a compound falling within Formulas I such as for example,peptides as depicted in SEQ ID NOs: 1-15, 20-26. The a urogenitaldisorder or a genitor-urological disorders can be renal disease; abladder disorder; disorders of the reproductive system; gynecologicdisorders; urinary tract disorder; incontinence; disorders of the male(spermatogenesis, spermatic motility), and female reproductive system;sexual dysfunction; erectile dysfunction; embryogenesis; and conditionsassociated with pregnancy. These are also used in pregnancy monitoring.As used herein, the term “conditions associated with pregnancy”includes, but is not limited to, conditions of fertilisation, pregnancy,parturition and lactation. The invention in another embodiment includesthe foregoing method, wherein said pregnancy related disorders areselected from a group consisting of Abnormal endometrial angiogenesis;Placental development defects; Cervical ripening (softening); Abnormalimplantation; Nipple development and disfunction; Pregnancy relatedremodeling of the Uterine tissue; Endometriosis; Preeclampsia; Lactationdisorders; Estrogenic and non-estrogenic related hormonal disorders;Pre-term labor; post term labor; and Labor complications.

Also provided by the invention is a method of prevention or treatment ofrespiratory diseases in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, II, IV and VI, such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The respiratory diseases can be asthma,bronchial disease, lung diseases, chronic obstructive pulmonary disease(COPD), Acute Respiratory Distress Syndrome (ARDS), severe acuterespiratory syndrome (SARS), Fibrosis related Asthma, cystic fibrosis.

Also provided by the invention is a method of prevention or treatment ofmetabolic disorders in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, such as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26. The metabolic disorders can be diabetes, diabetes mellitus,lipodystrophy, hyperthyroidism, glaucoma, hyperlipidaemia, non-insulindependent diabetes, Food intake; Water intake; Feeding and drinkingbehaviors, Anorexia, Cachexia (cancer and non cancer related); Fat andlipid metabolism; and Energy control, appetite control and obesity.

Also provided by the invention is a method of prevention and treatmentof kidney diseases in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinI, such as for example, peptides as depicted in SEQ ID NOs: 1-15, 20-26.The kidney diseases can be diabetic nephropathy; glomerulosclerosis;nephropathies; renal impairment; scleroderma renal crisis and chronicrenal failure.

Also provided by the invention is a method of prevention and treatmentof angiogenesis related conditions in a subject by administering to asubject in need thereof a therapeutically effective amount of a compoundfalling within Formulas I such as for example, peptides as depicted inSEQ ID NOs: 1-15, 20-26. The angiogenesis related conditions includingbut not limited to retinal angiogenesis in a number of human oculardiseases such as diabetes mellitus, retinopathy of prematury, andage-related macular degeneration, or cancer associated angiogenesis inprimary or metastatic cancer, including but not limited to cancer of theprostate, brain, breast, colorectal, lung, ovarian, pancreatic, renal,cervical, melanoma, soft tissue sarcomas, lymphomas, head-and-neck, andglioblastomas.

Also provided by the invention is a method of treating central nervoussystem (CNS) disorder, in a subject by administering to a subject inneed thereof a therapeutically effective amount of a compound fallingwithin Formulas I, such as for example, peptides as depicted in SEQ IDNOs: 1-15, 20-26. The central nervous system (CNS) disorder, includingbut not limited to central and peripheral degenerative neuropathies;neuroprotection; impaired cognition; anxiety disorders, pain control,food intake, a behavioral disorder, a learning disorder, a sleepdisorder, a memory disorder, a pathologic response to anesthesia,addiction, depression, migraine, a menstruation disorder, muscle spasm,opiate dependence, dementia, Alzheimer's disease, Parkinson's disease,cortical function, locomotor activity, Alcohol and Drug addiction andabuse; Impared memory; Feeding and drinking related behaviours; Stresscontrol, Bipolar disorder; Schyzophrenia; Schyzoaffective; MultipleSclerosis (MS); Stroke and stroke damage repair (Ischemia protection);Vasculature and re-vasculature in the brain; and Brain tissueregeneration and a peripheral nervous system disorder.

Also provided by the invention is a method of treating bone and bonerelated disorders in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, such as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26. The bone and bone related disorders including but notlimited to Osteoporosis; Osteoarthritis; Osteopetrosis; Boneinconsistency; Osteosarcoma; and Cancer matastesis to the bone.

Also provided by the invention is a method of treating endothelialdysfunction disease, in a subject by administering to a subject in needthereof a therapeutically effective amount of a compound falling withinFormulas I, such as for example, peptides as depicted in SEQ ID NOs:1-15, 20-26. The endothelial dysfunction disease is selected from agroup consisting of cardiovascular diseases, high blood pressure,atherosclerosis, thrombosis, myocardial infarct, heart failure, renaldiseases, plurimetabolic syndrome, erectile dysfunction; vasculitis; anddiseases of the central nervous system (CNS).

Optionally, the cDNA that encodes the peptide sequences of the inventionare used in gene therapy to treat the respective diseases, disordersand/or conditions, as detailed hereinabove.

The invention will be further illustrated in the following examples.

Example 1 Peptides Syntheses

All P74, P59-S and P59 related peptides derive from C1QT8, a Collagenrepeat containing Hypothetical protein (SEQ ID NO:19) or its orthologousor homologous sequences:

>P60827 C1QT8_HUMAN Complement C1q tumor necrosis factor-related protein8-Homo sapiens (Human).

MAAPALLLLALLLPVGAWPGLPRRPCVHCCRPAWPPGPYARVSDRDLWRGDLWRGLPRVRPTIDIEILKGEKGEAGVRGRAGRSGKEGPPGARGLQGRRGQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVGRREGLHSSDHFQAVPFDTELVNLDGAFDLAAGRFLCTVPGVYFLSLNVHTWNYKETYLHIMLNRRPAAVLYAQPSERSVMQAQSLMLLLAAGDAVWVRMFQRDRDNAIY GEHGDLYITFSGHLVKPAAELPeptide P59-S-Amide (Amide): (SEQ ID NO: 1) AYAAFSV-Amide; PeptideP59-SG (free acid Gly) (SEQ ID NO: 2) AYAAFSV; Peptide P59-Amide (amide)(SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; Peptide P59 (free acid)(SEQ ID NO: 4) GQKGQVGPPGAACRRAYAAFSV; Peptide P59C13V-Amide (amide)(SEQ ID NO: 5) GQKGQVGPPGAAV*RRAYAAFSV-Amide Peptide P59C13V (free acid)(SEQ ID NO: 6) GQKGQVGPPGAAV*RRAYAAFSV; Peptide P74-Amide (Amide): (SEQID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide Peptide P74 (free acid)(SEQ ID NO: 8) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV Peptide P74C13V (amide)(SEQ ID NO: 9) GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSV-Amide Peptide P74C13V(free acid) (SEQ ID NO: 10) GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSV PeptideP59SG (free acid Gly) (SEQ ID NO: 11) AYAAFSVG; Peptide P59-G (free acidGly) (SEQ ID NO: 12) GQKGQVGPPGAACRRAYAAFSVG; Peptide P59C13V-G (freeacid Gly) (SEQ ID NO: 13) GQKGQVGPPGAAV*RRAYAAFSVG; Peptide P74-G (freeacid Gly) (SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG PeptideP74C13V-G (free acid Gly) (SEQ ID NO: 15)GQKGQVGPPGAAV*RRAYAAFSVGRRAYAAFSVG Peptide P59-Chimpanzee (SEQ ID NO:20) GQKGQVGPPGAACQRAYAAFSVG; Peptide P59-Orangutan (SEQ ID NO: 21)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Rhesus (SEQ ID NO: 22)GQKGQVGPPGAPCQRAYAAFSVG; Peptide P59-Cow (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; Peptide P59-Chicken (SEQ ID NO: 24)GQKGQPGPQGHSCKQLYAAFSVG; Peptide P59-C1QTNF1 (Human) (SEQ ID NO: 25)GQKGSMGAPGERCKSHYAAFSVG; Peptide P59-Rat (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG. *The Cys residue within the peptides wasreplaced by V in the synthesized peptides made to prevent dimerizationat the cysteine residue in the peptides.

Peptides were synthesized by the solid phase peptide synthesis (SPPS)method, cleaved from the resin, and purified by RP-HPLC unless statedotherwise. The peptide's identity was verified by mass spectrometry.Final purity of peptide was >90% as measured by RP-HPLC. Peptides werediluted in PBS containing 0.1% BSA. All plates were stored at −80C untiluse.

Example 2 Effect of Peptides P59C13V (P59) (SEQ ID NO:6) and P74C13V(P74) (SEQ ID NO:10) on Cyclic Amp Accumulation in Cho-K1 CellsExpressing LGR7 and LGR8

The ability of Peptides to affect cAMP concentration was examined inCHO-K1 cells transiently transfected with LGR7 (RXFP1) or LGR8 (RXFP2).The experiment measured cAMP concentration in response to the additionof the peptides.

LGR7 (RXFP1) or LGR8 (RXFP2) were transiently transfected into CHO-K1cells as follows: Cells (12 million) were plated into T75 flasks on theday preceding transfection. Cells were transfected with a GPCR DNA andG_(α,16) using a lipid technique according to the manufacturer'srecommendation. Cells were transfected for 5 hours, then re-plated into96-well dishes (60,000 cells per well) and grown overnight. Transfectedcells were plated into 24 wells of a 96-well plate. Cells werepre-treated with 0.5 mM IBMX (stimulation buffer) for 10 min at 37° C.,then stimulated with either a positive control (Relaxin 2) or acandidate peptide (for Gs functional examination), followed bystimulation or a preincubation with 10 μM forskolin (for Gi functionalexamination). Following a 20 minutes stimulation by incubation with 1 uMof either positive control or the tested peptides, either with orwithout forskolin, intracellular cAMP was assayed using the Hit HuntercAMP kit (DiscoveRx Corporation) according to the manufacturer'srecommended protocol. Data was converted to nmol of cAMP by running astandard cAMP curve.

Relaxin was used as a positive control. cAMP level was elevated bypre-treatment with forskolin.

The transfected CHO-K1 cells were treated with 10 uM of Forskolin(Applied cell sciences (ACS)) for 10 minutes and then challenged with 1uM of H2 Relaxin (ACS) (as a positive control), 1 uM of Peptide P59C13V(P59) (SEQ ID NO: 6), and Peptide P74C13V (P74) (SEQ ID NO: 10).

The results are shown in FIG. 1. As can be seen from FIG. 1, bothpeptides P59C13V (P59) (SEQ ID NO: 6) and P74C13V (SEQ ID NO: 10) showeda cAMP inhibitory effect with respect to stimulation by Forskolin, andin relation to the effect H2 Relaxin positive control had on the cells.A clear cAMP inhibition (Gi) effect was demonstrated by P59C13V (P59)(SEQ ID NO: 6) and P74C13V (P74) (SEQ ID NO: 10) on both receptors at 1uM. Relaxin affected only the LGR7 (RXFP1) receptor. P59C13V (P59) (SEQID NO: 6) showed a stronger effect, than P74C13V (P74) (SEQ ID NO: 10),however, both peptides showed stronger effect when compared to thereduction in cAMP with the positive control (Relaxin 2).

A dose response effect of P59C13V (P59) and P74C13V (P74) on CHO-K1cells transiently transfected with LGR8 (RXFP2) was further examined(Method as above). CHO-K1 cells transiently transfected with LGR8(RXFP2) were treated with 20 uM of Forskolin for 10 minutes and thenchallenged with either INSL3 (as a positive control), P59C13V (SEQ IDNO: 6) or P74C13V (P74) (SEQ ID NO: 10) at increasing concentrationsfrom 1 nM to 10 uM. The results are shown in FIG. 2. As can be seen fromFIG. 2, P59C13V (P59) (SEQ ID NO: 10) showed a slight decrease in cAMPat concentrations lower than 100 nM; (Gi effect) and a slight increase(Gs effect) at concentrations higher than 100 nM, as compared to INSL3.INSL3 showed a slight dose dependent increase in lower concentrations(<100 nM; Gs effect), and a sharp decrease (Gi effect) in higherconcentrations (>100 nM). P74C13V (P74) (SEQ ID NO: 10) showed a patternsimilar to INSL3 at lower concentrations (slight increase in cAMP) butno effect at higher concentrations.

Example 3 Effect of Peptides P59C13V (P59) (SEQ ID NO: 6) and P74C13V(P74) (SEQ ID NO: 10) on cAMP Accumulation in CHO-K1 Cells ExpressingLGR4, LGR5 or LGR6

The ability of Peptides to affect cAMP concentration was examined inCHO-K1 cells transiently transfected the GPCR's LGR4, LGR5, or LGR6 andtreated with 1 uM of P59C13V (P59) (SEQ ID NO: 6), P74C13V (P74) ((SEQID NO: 10), or HCG (ACS) which was used as a positive control. cAMPaccumulation in the cells was then measured as described above with (Gi)or without (Gs) pre-treatment with Forskolin.

The results are shown in FIG. 3. According to FIG. 3A, a significantincrease in cAMP accumulation was demonstrated for LGR6 transfectedcells when challenged with P74C13V (P74) (SEQ ID NO: 10). No significantGs activation was found for any of the peptides on any of the other LGRtransfected cells.

According to FIG. 3B, a significant Gi effect (decrease in cAMPaccumulation) was demonstrated for LGR5 transfected cells whenchallenged with P59C13V (P59) (SEQ ID NO: 6) or P74C13V (P74) (SEQ IDNO: 10). No significant Gi activation was found for the positive control(HCG).

According to FIG. 3C, a slight yet statistically significant Gi effect(decrease in cAMP accumulation) was demonstrated for LGR4 transfectedcells when challenged with P59C13V (P59) (SEQ ID NO: 6) or P74C13V (P74)(SEQ ID NO: 10). A slight but significant Gi activation was found forthe positive control (HCG).

FIG. 3 (A-C) indicate a slight Gs activation of LGR6 by P74C13V (P74), aslight Gi activation of LGR5 by both P59C13V (P59) (SEQ ID NO: 6) andP74C13V (P74) (SEQ ID NO: 10) and a slight Gi activation of LGR4 by bothP59C13V (P59) (SEQ ID NO: 6) and P74C13V (P74) (SEQ ID NO: 10) as wellas HCG.

Example 4 Dose Response Stimulation of Camp Mediated by Peptides P59(SEQ ID NO: 6) and P74 (SEQ ID NO: 10) in CHO-K1 Cells Expressing LGR7

CHO-K1 cells transiently transfected with LGR7 (RXFP1) were treated with10 uM of Forskolin for 10 minutes and then challenged with either H2Relaxin (as a positive control), P59C13V (P59) (SEQ ID NO: 6) or P74C13V(P74) (SEQ ID NO: 10) at increasing concentrations, from 1 nm to 10 uM(FIG. 4A) and from 3 nM to 100 nM (FIG. 4B). cAMP concentrations weredetermined at each point, as described above with (FIG. 4A) or without(FIG. 4B) the presence of Forskolin.

The results are presented in FIG. 4. As can be seen from FIG. 4A,P59C13V (SEQ ID NO: 6) showed a bell-shaped curve as in lowerconcentrations there was an increase in cAMP (Gs effect), in the 30 and100 nM doses, as compared to H2 Relaxin, whereas Relaxin showed a dosedependent decrease (Gi) effect in higher concentrations (>100 nM).P74C13V (SEQ ID NO: 10) showed no significant effect in this assay.

FIG. 4B describes a Gs (cAMP stimulation and increase in cAMPaccumulation) dose response was measured for LGR7 (RXFP1)-transfectedCHO-K1 cells using low concentrations (3-100 nM) of P59C13V (P59) (SEQID NO: 6) or H2 Relaxin. Both P59C13V (P59) and H2 Relaxin showed a dosedependent increase in cAMP. As shown in FIG. 4B, P59C13V (P59) (SEQ IDNO: 6) was more effective than Relaxin in increasing cAMP levels inthese studies.

The ability of P59C13V (SEQ ID NO: 6) and H3 Relaxin to activate CHO-K1cells transfected by GPR135 (RXFP3), which is a non-LGR, Relaxin relatedreceptor, was tested. The assay was performed by Euroscreen. For cAMPconcentration analysis, Euroscreen cat. ES-656-A-cells (RXFP3,Euroscreen cat. ES-656-A) grown to mid-log phase in culture mediawithout antibiotics were detached with PBS-EDTA, centrifuged andresuspended in assay buffer at a concentration of 7.5×105 cells/ml. Thetest was performed in 96 well plates. For agonist testing, 12 μl ofcells (5×103 cells/well) were mixed with 12 μl of agonist EitherH3-Relaxin or P59C13V (P59) (SEQ ID NO: 6) at increasing concentrations.The plates were then incubated for 30 min at room temperature. Afteraddition of the lysis buffer, cAMP concentrations were estimated,according to the manufacturer specification, with the HTRF kit fromCis-Bio International (cat n° 62AM2PEB).

Relaxin 3 Fit (Eeuroscreen) was used as a positive control. RXFP3(GPCR135) transfected cells were challenged with increasingconcentrations of Relaxin 3 (H3 Relaxin—as a positive control—1 pM-1 uM)or P59C13V (P59) (SEQ ID NO: 6) (1 nM-3 uM). The results are shown inFIG. 5. As can be seen from FIG. 5, P59C13V (P59) (SEQ ID NO: 6) didn'tshow any cAMP activation (Gs) of RXFP3, while a strong cAMP stimulationwas demonstrated by H3 Relaxin.

In this experiment the P59C13V (P59) (SEQ ID NO: 6) did not activate acAMP stimulation effect on RXFP3 transfected CHO-K1 cells. However,without wishing to be bound by theory, the P59C13V (P59) (SEQ ID NO: 6)peptide can either bind or activate a different pathway when applied onRXFP3 transfected cells. This experiment does not rule out thepossibility that the P59C13V (P59) (SEQ ID NO: 6) might activate a Gi ora different G-protein mediated effect on RXFP3 receptor, nor determinethe effect of and of the other peptides on this receptor (i.e. SEQ. ID.No. 1-5 or 7-15).

Example 5 Activity of P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) inCells Expressing LGR7 or LGR8 Together with pCRE-β-GAL

In order to further test the activity of P59C13V (P59) (SEQ ID NO: 6),P59S-Amide (P59S) (SEQ ID NO: 1), P59C13V-Amide (P59Amide) (SEQ ID NO:5) and P74C13V (P74) (SEQ ID NO: 10), the following assay was performed.The assay was done to assess the ability of P59C13V (P59) (SEQ ID NO:6), P59S-Amide (P59S) (SEQ ID NO: 1), P59C13V-Amide (P59Amide) (SEQ IDNO: 5) and P74C13V (P74) (SEQ ID NO: 10) peptides to replace H2 Relaxinor INSL3 activity in stable HEK-293T cell lines expressing the LGR7(RXFP1) or the LGR8 (RXFP2) receptors, respectively, by assessment ofthe ability of these peptides to increase cAMP activity in cell linesexpressing LGR7 (RXFP1) or LGR8 (RXFP2) respectively, together withpCRE-β-gal. The pCRE-β-gal assay is a standard and well establishedassay at the Howard Florey Institute (Melbourne, Australia) (Halls M L,Bathgate R A, Summers R J.—Comparison of signaling pathways activated bythe relaxin family peptide receptors, RXFP1 and RXFP2, using reportergenes. J Pharmacol Exp Ther. 2007 January; 320(1):281-90.)

The ability of P59C13V (P59) (SEQ ID NO: 6), P59S-Amide (P59S) (SEQ IDNO: 1), P59C13V-Amide (P59Amide) (SEQ ID NO: 5) and P74C13V (P74) (SEQID NO: 10) to influence cAMP activity in HEK-293T LGR7/pCRE-β-gal stableor LGR8/pCRE-β-gal transfected cells was tested in parallel to H2relaxin and INSL3 respectively, according to the protocol described inHalls M L, et al., J Pharmacol Exp Ther. 2007 January; 320(1):281-90.

The experiment was repeated at least three times. The followingconcentrations of each peptide were tested: 0.01 nM, 0.1 nM, 1 nM, 10nM, 100 nM, 1 μM.

No activation was shown for any of the peptides on LGR7/pCRE-β-galtransfected HEK-293T cells (data not shown).

The results demonstrating activation of LGR8 by P74C13V (SEQ ID NO: 10),P59C13V with (P59-Amide) (SEQ ID NO: 5) and without (P59) amide (SEQ IDNO: 6) and P59S-Amide (SEQ ID NO: 1), are shown in FIGS. 6A and 6B. Theresults are presented as a percentage of INSL3 activity inLGR8/pCRE-β-gal transfected HEK-293T cells as a function of theconcentration (Represented in Log [M]).

FIG. 6A demonstrates a bell shaped response of LGR8/pCRE-β-galtransfected HEK-293T cells to P74C13V (SEQ ID NO: 10) in all three ofthe assays. The P59S-Amide (SEQ ID NO: 1) effect was less pronounced.FIG. 6B shows that P59C13V (P59) (SEQ ID NO: 6) demonstrates a weakactivation effect (˜25% of INSL3) on LGR8/pCRE-β-gal transfectedHEK-293T cells, while P59C13V-amide (P59-Amide) (SEQ ID NO: 5) had avery weak effect.

The ability of P74C13V (P74) (SEQ ID NO: 10) and P59C13V (P59) andP59C13V-Amide (P59-Amide) (SEQ ID NO: 6 and 5 respectively) to activateLGR7 (RXFP1) and LGR8 (RXFP2) was further tested by testing the abilityof these peptides to influence cAMP activity induced by 5 μM Forskolinin CHO-K1 cells transiently transfected with LGR7/pCRE-β-gal orLGR8/pCRE-β-gal as compared to H2 relaxin and INSL3 respectively.

LGR7 transfected CHO-K1 cells were treated with 5 uM of Forskilin tostimulate cAMP. The cells were then challenged with increasing doses ofH2 Relaxin (as positive control), P74C13V (P74) (SEQ ID NO: 10), P59C13Vwith (P59-Amide) and without (P59) amide and P59S-Amide (P59S) (SEQ IDNO: 5, 6 and 1, respectively). The results demonstrating the effect ofP74C13V (P74) (SEQ ID NO: 10), P59C13V with (P59-Amide) and without(P59) amide (SEQ ID NO: 5 and 6, respectively) and P59S-Amide (SEQ IDNO: 1) on LGR7/pCRE-β-gal transiently transfected CHO-K1 cells ispresented in FIGS. 7A and 7B. The results are presented as a percentageof Forskolin activity at increasing concentrations of each peptide(represented in log [M]). The baseline for the experiment was theactivity of Forskolin alone (100%). Each point represents threeunrelated repeats for each experiment together with their standard errorbars.

FIG. 7A presents a bell-shaped activation pattern shown by P74C13V (P74)(SEQ ID NO: 10) on LGR7/pCRE-β-gal transiently transfected CHO-K1 cells.The activation of P74C13V (P74) (SEQ ID NO: 10) seems to be strongerthan that of H2 Relaxin at the lower concentrations (as was alreadydemonstrated in FIGS. 4A and 4B), whereas H2 Relaxin showed an increasedactivation at higher concentrations. P59S-Amide (SEQ ID NO: 1) showed amild activation (of only ˜X2 or Forskolin) at higher concentrations only(100 nM and 1-10 uM)

FIG. 7B presents a dual activation pattern effect, demonstrating anactivation peak at lower concentrations and second peak in cAMPactivation at higher concentrations, shown by P59C13V (P59) (SEQ ID NO:6); and a slight activation pattern shown by P59C13V-amide (P59-Amide)(SEQ ID NO: 5) on LGR7/pCRE-β-gal transiently transfected CHO-K1 cells.

LGR8 transfected CHO-K1 cells were treated with 5 uM of Forskilin tostimulate cAMP. The cells were then challenged with increasing doses ofINSL3 (as positive control), P74C13V (P74) (SEQ ID NO: 10), P59C13V with(P59-Amide) and without (P59) amide (SEQ ID NO: 5 and 6, respectively)and P59S-Amide (P59-S) (SEQ ID NO: 1). The results demonstrating theeffect of P74C13V (P74) (SEQ ID NO: 10), P59C13V with (P59-Amide) andwithout (P59) amide (SEQ ID NO: 5 and 6, respectively) and P59S-Amide(P59S-Amide) (SEQ ID NO: 1) on LGR8/pCRE-β-gal transiently transfectedCHO-K1 cells is presented in FIGS. 8A and 8B. The results are presentedas a percentage of Forskolin activity at various concentrations of thepeptide (logarithmic scale). Each point represents three unrelatedrepeats for each experiment.

FIG. 8A presents a bell-shaped activation pattern, shown by P74C13V(P74) (SEQ ID NO: 10) similar to that of INSL3 at lower concentrationson LGR8/pCRE-β-gal transiently transfected CHO-K1 cells. The activationof P74C13V (P74) (SEQ ID NO: 10) seems to be similar yet slightly weakerthan that or INSL3 at the lower concentrations (as was alreadydemonstrated in FIG. 7A for LGR7), whereas INSL3 showed an increasedactivation at higher concentrations. P59S-Amide (SEQ ID NO: 1) showed amild activation (of only ˜X2 or Forskolin) at higher concentrations only(100 nM and 1-10 uM).

FIG. 8B presents a dual activation pattern effect, demonstrating a peakin activation at lower concentrations and second increase in cAMPactivation at higher concentrations shown by P59C13V (P59) (SEQ ID NO:6); and a slight activation pattern shown by P59C13V-amide (P59-Amide)(SEQ ID NO: 5) on LGR8/pCRE-β-gal transiently transfected CHO-K1 cells.

Example 6 Competition Assays of P74C13V (SEQ ID NO: 10) and P59C13V (SEQID NO: 6) with Europium Labelled H2 Relaxin or INSL3 in Cells ExpressingLGR7 or LGR8

The ability of Peptides P74C13V (SEQ ID NO: 10) and P59C13V (SEQ ID NO:6) to compete with the binding of known ligands to LGR7 or LGR8 wastested by testing the ability of P74C13V (SEQ ID NO: 10) and P59C13V(SEQ ID NO: 6) to compete with the binding of Europium labelled H2Relaxin or INSL3 (manufactured by the H. Florey institute) to stableHEK-293T cell lines expressing LGR7 or LGR8 respectively.

HEK-293T cell lines stabled expressing LGR7 (RXFP1) were treated withEuropium labelled H2 Relaxin (manufactured by the H. Florey institute)The cells were then treated with decimal increasing concentrations (0.01nM, 0.1 nM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM) of un-labeled H2 Relaxin(as a positive control) and the tested peptides P59C13V (P59) (SEQ IDNO: 6), P59C13V-Amide (P59-Amide) (SEQ ID NO: 5), P59-S-Amide (P59S)(SEQ ID NO: 1) and P74C13V (P74) (SEQ ID NO: 10). The competitionresults of three independent experiments are shown in FIG. 9. Thebinding results are shown as a percentage of specific binding of theradioactive test H2 Relaxin (where 100% is the baseline radiationlevel). As can be seen from FIG. 9, no competition was shown for thebinding of Europium-H2 Relaxin by any of the tested peptides. Withoutwishing to be bound by a single theory, the activity of peptides P59C13V(SEQ ID NO: 6), P59C13V-Amide (SEQ ID NO: 5), P59S-Amide (SEQ ID NO: 1)and P74C13V (SEQ ID NO: 10) on LGR7 (RXFP1), might be mediated bybinding of the peptides to a different binding site, since the bindingof the above peptides does not interfere, compete or affect the bindingof the Europium H2 Relaxin to LGR7.

HEK-293T cell lines expressing LGR8 (RXFP2) were treated with Europiumlabeled INSL3 (manufactured by the H. Florey institute). The cells werethen treated with decimal increasing concentrations (0.01 nM, 0.1 nM, 1nM, 10 nM, 100 nM, 1 μM, 10 μM) of un-labeled INSL3 (as a positivecontrol) and the tested peptides P59C13V (P59) (SEQ ID NO: 6),P59C13V-Amide (P59-Amide) (SEQ ID NO: 5), P59-S-Amide (P59S) (SEQ IDNO: 1) and P74C13V (P74) (SEQ ID NO: 10). The competition results ofthree independent experiments are shown in FIG. 10. The binding resultsare shown as a percentage of specific binding of the radioactive testINSL3 (where 100% is the baseline radiation level). As can be seen fromFIG. 10, P59C13V (P59) (SEQ ID NO: 6) demonstrates a positivecooperative or allosteric effect. showing ˜125% of specific binding at 1nM and higher concentrations. This means that the binding affinity ofthe Eur-INSL3 was increased by binding/activity of the P59C13V (P59)(SEQ ID NO: 6) peptide on the LGR8 receptor. The results with the otherpeptides (P59C13V-amide (P59-Amide) (SEQ ID NO: 5), P59-S-Amide (P59S)(SEQ ID NO: 1) and P74C13V (P74) (SEQ ID NO: 10)) were less apparent butall four peptides showed alteration and competition/alosteric effect onthe binding of Europium-INSL3 on LGR8 (RXFP2). Without wishing to bebound by a single theory, the binding of the peptides to the LGR8receptor, probably through a different binding site than that of INSL3,there is an allosteric effect that might alter the affinity of thereceptor to the INSL3 ligand, might suggesting a synergistic effect ofthe above peptides and INSL3 on the activity of the LGR8 (RXFP2)receptor.

Example 7 Real Time Activation Assay Tested by ACEA Cell ImpedanceSystem Method for Analysis of Peptides' Ability to ActivateRelaxin-Related Family of Receptors Using ACEA RT-CES Screen ACEA RT-CESScreen Background:

ACEA RT-CES screen is a noninvasive and label-free assay for GPCRs thatcan be used with both engineered and nonengineered cell lines. The assayis based on using cell-electrode impedance to measure minute changes incellular morphology as a result of ligand-dependent GPCR activation andis described in Naichen Yu, et al., Anal. Chem., 78 (1), 35-43, 2006.10.1021/ac051695v S0003-2700(05)01695-1. The Rho family of smallGTPases, which include Rho, Rac, and CDC42, are well-characterizedeffectors of oncogenes, growth factor and adhesion-mediated signalingpathways and are not classically thought of as being key effectors forGPCRs. Rho family GTPases participate in a number of cellular processes,the main one being regulation and maintenance of specific structureswithin the actin cytoskeleton framework. GPCRs have been shown tomodulate the actin cytoskeleton and hence cell morphology in a veryspecific manner depending on the Rho family GTPase being activated. Thecurrent view of the actin cytoskeleton is that of a dynamic and plasticsystem that is a reflection and manifestation of the intracellularsignaling and not simply a static structure designed to maintaincellular architecture. Since GPCRs couple to the actin cytoskeletalnetwork and induce very defined morphological changes, it is possible toharness this information as a functional and biologically relevantreadout for GPCRs.

ACEA biosciences, has designed electronic cell sensor arrays embedded inthe bottom of the well of microtiter plates that are capable ofmeasuring minute changes in cell morphology. The electronic sensorsmeasure changes in cell-substrate impedance as a result of thedisruption of the ionic environment due to the presence of cell and cellmorphology dynamics. The main advantages offered by using cell-substrateimpedance and cell morphology as a readout are that both exogenouslyexpressed and endogenous receptors can be assayed without the need forengineering the cell with promiscuous G proteins and reporters orlabeling the cells with dyes. In addition, since the readout isnoninvasive, multiple stimulations with the same ligand or differentligands can be performed to assess events such as desensitization andreceptor cross-talk. Finally, another major aspect of usingcell-substrate impedance and cell morphology as a readout is thatpotentially all GPCRs, regardless of the signaling pathways, can befunctionally monitored.

ACEA RT-CES Screen Experimental Procedure & Protocol

Peptides were synthesized by the solid phase peptide synthesis (SPPS)method, cleaved from the resin, and purified by RP-HPLC unless statedotherwise. The peptide's identity was verified by mass spectrometry.Final purity of peptide was >90% as measured by RP-HPLC. Peptides werediluted in Water (DDW—high purity) containing 0.1% BSA. In some casespeptides were dissolved in Tris-HCl buffer (pH8.2). In other caseseither the addition of 1% DMSO or s brief sonication was required. Allplates were stored at −80C until use.

Cells: CHOk1 cell-line (ATCC CCL-61). Cells were maintained andpropagated in F-12 HAM nutrient mix (Gibco. Cat#21765-029) supplementedwith 10% HI-FBS (Biological Ind. Cat#04-121-1), 2.5 mL of 200 mmL-Glutamine in Saline Solution (Biological Ind. Cat#03-020-1), 5 mL ofPenicillin-Streptomycin Solution 10000/mLPenicillin G & 10 mg/mLStreptomycin Sulfate (Biological Ind. Cat#03-031-1).

Subculturing Cells:

Cells were freshly thawed from liquid N2 and were sub-cultured 1:10twice a week according to the following protocol:

Remove and discard culture medium.

Briefly rinse the cell layer with 0.25% (w/v) Trypsin-0.53 mM EDTAsolution to remove all traces of serum which contains trypsin inhibitor.

Incubate for 5 minutes at 37° C. in CO2 humidified incubator.

Examine under the microscope until cell layer is dispersed.

Add 10 to 12 mL of complete growth medium and aspirate cells by gentlypipetting.

Take a 300 μl sample from the cell suspension and count concentration,viability and aggregate rate using the CEDEX conuter.

Centrifuge cells for 6 minutes at 1200 rpm.

Re-suspend cells in complete growth medium to the desirable cellconcentration.

DNA Transfection:

All transfections were performed using the FUGENE6 reagent (Roche.Cat#11-814-443-001, Expiry date: May 2008).

Transfection Protocol:

One day before the transfection 2×106 CHO-k1 cells were plated in T75flask containing 10 mL of complete growth medium. On the transfectiondate, FUGENE6 transfection reagent was warmed to ambient temperature by15 minutes incubation at room temperature and mixed prior to use byvortex.

The following was diluted in a sterile microfuge tube: 50 μl of FUGENE6into 650 μl of serum free F12-HAM medium and tubes were incubated for 10min at RT.

The transfected DNA mix was prepared in another sterile microfuge tube;Total of 20 μg plasmid DNA mixture which is composed of 18 μg of thetarget GPCR plasmid and of 2 μg of pIRES plasmid carrying eGFP reportergene.

The plasmids DNA mixture was then added to the transfection reagent tubeand incubated 35 min at RT with gently tapping once every 5 minutes tomix the contents. The DNA-FuGene complex was added drop wise to flaskand spread by gentle swirling.

The transfected cells were kept for 24 hours at 37° C. in CO2 humidifiedincubator and monitored under fluorescent microscope for the detectionof green light omitted from GFP transfected cells.

ACEA RT-CES Protocol; Screening for GPCR Activating Peptides:

In this protocol the RCD96 E-plate device (ACEA Biosciences Inc.) wereused for seeding, monitoring and activating the CHO-k1 transfectedcells.

Pre-treatment E-Plates: The E-plate wells were coated with 100 uL of 1mg/ml Gelatine (Fluka. Cat#48720). The gelatine was dissolved at 8 mg/mLin sterile DDW and was diluted 1:8 in DDW before added to the E-platewells. The plates covered with gelatine were incubated for 40 minutes at37° C. and 5% CO2 in a humidified atmosphere.

100 μl of sterile water were added and all liquids were removed. Theplates were washed twice more, now with 200 μl of sterile water andplate was taken for cells seeding

Cells Seeding:

Before the transfected cells were seeded, 80 ml SFM F12-HAM medium wereadded to each well of the E-plates and background levels were recorded.24-26 hours past transfection, medium was aspirated and cells weretrypsinized with 0.25% (w/v) Trypsin/EDTA solution and counted on theCEDEX counter.

Total cell number was calculated and cells were re-suspended in completegrowth medium to cell concentration ranging from 0.3125×106/mL up to0.4375×106/mL. From that dilution, 80 μl of cell suspension were seeded,resulting in 25,000 up to 35000 total cells in 5% FCS—complete growthmedium per each E-plate well.

Peptide Challenging:

22-26 hours past seeding (46-52 hours past transfection) and withconcordance to the recorded Cell Index (CI) the cells were prepared forthe addition of the activating peptides. Prior to adding the peptides,the complete medium was aspirated and replaced with 120 ml 37° C.pre-warmed SFM F12-HAM nutrient mixture.

1 hour later or when the CI reads are stabilized, the E-plate wasremoved from the 96X E-Plate Station for peptide challenge. Challengingwith the peptides was performed in duplicates by adding 5 μl of peptidesolution from an already made 250 μM daughter stock plates, resulting infinal peptide concentration of 10 μM. The peptide solution contains thepeptide dissolved in DDW supplemented with 0.1% Albumin (Sigma-aldrichCat# A3059, Fraction V, ˜99%, Essentially γ-globulin free).

The screening was done on transiently transfected CHO-K1 cells and wasdone in two stages:

-   -   1. Screening phase—Challenging the relaxin-related family of        receptors transfected CHO-K1 cells with 10 uM of each screened        peptide, using Calcitonin (1 uM) as an assay internal positive        control (the receptor for Calcitonin is endogenously expressed        by CHO-K1 cells), 0.1% BSA as a negative control. 1 uM of H2        Relaxin was used as a positive control for the transfected        receptor.    -   2. Dose response phase—Challenging the LGR7 transfected CHO-K1        with 0.03 uM, 0.1 uM, 0.3 uM, 1 uM, 3 uM and 10 uM of each        screened peptide, using H2 Relaxin as a positive control for the        transfected receptor.

Example 7-1 P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) MediatedActivation of the LGR-Related Family of Receptors in ACEA Cell ImpedanceDevice-Screening Phase

The ability of Peptides P74C13V (P74) (SEQ ID NO: 10) and P59C13V (P59)(SEQ ID NO: 6) to activate LGR7 (RXFP1) and LGR8 (RXFP2) receptors waschecked as described above in ACEA cell impedance device. The followingresults were obtained:

First the ability of Peptide P74C13V (SEQ ID NO: 10) (at 10 uM) and H2Relaxin (at 1 uM) to cause changes in cell impedence was measured inuntransfected CHO-K1 cells. The results are presented in FIG. 11. Cellindex was normalized to time point T1 (after peptide administration),marked in FIG. 11 by a vertical solid line (The dashed Vertical lineindicates the 20^(th) hour from the beginning of the experiment).Calcitonin was used as an internal positive control to evaluate thevalidity of the assay, as the calcitonin receptor is endogenouslyexpressed on CHO-K1 cells. BSA 0.1% was used as a negative control. Ascan be seen from FIG. 11, both H2 Relaxin and peptide P74C13V (P74)demonstrated a moderate effect on untransfected CHO-K1 cells. Withoutwishing to be bound by a single theory, the conclusion from this resultis that even though not detected at standard procedures, either the LGR7or LGR8 receptors are expressed, even if in a residual amount, by theCHO-K1 cell line.

Next, the ability of Peptide P74C13V (P74) (SEQ ID NO: 10) (at 10 uM)and H2 Relaxin (at 1 uM) to cause changes in cell impedence was measuredin CHO-K1 cells transfected with a non-relaxin related GPCR: GPR39(GPR39Human in SwissProt accession: 043194). The results are presentedin FIG. 12. Cell index was normalized to time point T1 (after peptideadministration), marked in FIG. 12 by a vertical solid line (the dashedvertical line indicates the 24^(th) hour from the beginning of theexperiment). Calcitonin was used as a positive control and BSA 0.1% wasused as a negative control. As can be seen from FIG. 12, both H2 Relaxinand peptide P74C13V (P74) (SEQ ID NO: 10) demonstrated a moderate yetvisible effect on GPR39 transfected CHO-K1 cells.

From these experiments, it can be seen that the transfection processitself was not the cause for the response presented above (in FIG. 11).

Next, the ability of Peptide P74C13V (P74) (SEQ ID NO: 10) (at 10 uM)and H2 Relaxin (at 1 uM) to cause changes in cell impedence was measuredin CHO-K1 cells transfected with a LGR7 (RXFP1). The results arepresented in FIG. 13. Cell index was normalized to time point T1 (afterpeptide administration), marked in FIG. 13 by a vertical solid line.Calcitonin was used as a positive control and BSA 0.1% was used as anegative control. As can be seen from FIG. 13, both H2 Relaxin andpeptide P74C13V (SEQ ID NO10) demonstrated a cellular cell impedance (asshown by the ACEA measurement) effect on LGR7 transfected CHO-K1 cells.The effect of P74C13V (SEQ ID NO: 10) was stronger than the effect of H2Relaxin, however, both peptides (H2 Relaxin and P74C13V (SEQ ID NO10))were found to be capable of activating a cellular response in LGR7transfected CHO-K1 cells.

The ability of Peptides P74C13V (P74) (SEQ ID NO: 10) (at 10 uM),P59C13V (P59) (SEQ ID NO: 6) (at 10 uM) and H2 Relaxin (at 1 uM) tocause changes in cell impedence was further measured in CHO-K1 cellstransfected with a LGR8 (RXFP2). The results are presented in FIG. 14.As above, cell index was normalized to time point T1 (after peptideadministration), marked in FIG. 14 by a vertical solid line (the dashedVertical line indicates the 23^(rd) hour from the beginning of theexperiment). As above, Calcitonin was used as a positive control and BSA0.1% was used as a negative control. As can be seen from FIG. 14, bothH2 Relaxin and peptide P74C13V (SEQ ID NO: 10) demonstrated a strongcellular effect on LGR8 transfected CHO-K1 cells. P59C13V (P59) (SEQ IDNO: 6) showed no significant effect as compared to BSA. From theseresults it seems that both peptides (H2 Relaxin and P74C13V (SEQ ID NO:10)) are capable of activating a cellular response in LGR8 transfectedCHO-K1 cells, however, no effect of P59C13V (P59) (SEQ ID NO: 6) on LGR8transfected CHO-K1 cells was demonstrated in this experiment. This couldbe explained by technical problem, such as degradation of the P59peptide in this experiment.

The ability of Peptide P74C13V (P74) (SEQ ID NO: 10) (10 uM) and H2Relaxin (1 uM) to cause changes in cell impedance was further measuredin CHO-K1 cells transfected with a LGR4. The results are presented inFIG. 15. As above, cell index was normalized to time point T1 (afterpeptide administration), marked in FIG. 15 by a vertical solid line. Asabove, Calcitonin was used as a positive control to and BSA 0.1% wasused as a negative control. As can be seen from FIG. 15, peptide P74C13V(SEQ ID NO: 10) demonstrated a moderate effect on LGR4 transfectedCHO-K1 cells. H2 Relaxin demonstrated a much stronger effect in thisassay. Without wishing to be bound by a single theory, this result maysuggest that H2 Relaxin is capable of binding and activating a cellularresponse though activation of the LGR4 receptor in LGR4 transfectedCHO-K1 cells, probably mediated by a non cAMP related pathway.

Example 7-1 P74 (SEQ ID NO: 10) and P59 (SEQ ID NO: 6) MediatedActivation of Relaxin-Related Family of Receptors in ACEA Cell ImpedanceDevice-Dose Response

Dose response activation testing of both (H2) Relaxin and P74C13V (P74)(SEQ ID NO: 10) in untransfected CHO-K1 cells showed no dose dependentactivation but only a moderate activation of the highest concentrations(i.e. 10 uM for P74C13V and 1 uM for H2 Relaxin) (data not shown).

The experiment was done similarly to the ACEA protocol above. Eachpeptide was added in triplicates at increasing concentrations (presentedin Log [M] on FIGS. 16B, 17B, 18B and 19B). Cell impedance was tested asdescribed and cell index was normalized to the time of peptide addition(T1—left vertical line in FIGS. 16A, 17A, 18A and 19A). Dose responsewas examined at end point of the experiment (one time point—indicated bythe right vertical line on FIGS. 16A-19A).

FIG. 16A presents a normalized cell index and a dose response curve forLGR7 (RXFP1) transfected CHO-K1 cells challenged by increasingconcentrations of H2 Relaxin (the dashed Vertical line indicates the25^(th) hour from the beginning of the experiment). The H2 Relaxinconcentrations tested were 0.16 nM; 0.8 nM; 4 nM; 20 nM; 100 nM; 500 nM.As shown in FIG. 16A, there is a clear dose response for the higherconcentrations, with an estimated EC50 of ˜100 nM.

FIG. 16B presents a normalized cell index and a dose response curve forLGR7 transfected CHO-K1 cells challenged by increasing concentrations ofH2 Relaxin at time point 25.5 hours (vertical right line in FIG. 16A). Adose response is presented as a normalized cell index per Log ofconcentration (M). As demonstrated in FIG. 16B, a clear dose dependentactivation of LGR7 by H2 relaxin was found.

FIG. 17A presents a normalized cell index and a dose response curve forLGR7 (RXFP1) transfected CHO-K1 cells challenged by increasingconcentrations of peptide P74C13V (P74) (SEQ ID NO:10) (the dashedVertical lines indicate the 25^(th) and 26^(th) hours from the beginningof the experiment). The P74C13V (P74) (SEQ ID NO: 10) concentrationstested were 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; 10 uM. As shown inFIG. 17A, there is a clear dose response for the high concentrations,with an estimated EC50 of ˜300 nM.

FIG. 17B presents a normalized cell index and a dose response curve forLGR7 transfected CHO-K1 cells challenged by increasing concentrations ofpeptide P74C13V (SEQ ID NO:10) at time point 26.5 hours (vertical rightline in FIG. 17A). A dose response is presented as a normalized cellindex per Log of concentration (M). As can be seen from FIG. 17B a cleardose dependent activation of LGR7 by P74C13V (SEQ ID NO:10) was found.The affinity found for P74C13V (SEQ ID NO:10) to LGR7 was lower thanthat of H2 Relaxin.

FIG. 18A presents a normalized cell index and a dose response curve forLGR7 (RXFP1) transfected CHO-K1 cells challenged by increasingconcentrations of peptide P59C13V (P59) (SEQ ID NO: 6) (the dashedVertical lines indicate the 25^(th) and 26^(th) hours from the beginningof the experiment). The P59C13V (P59) (SEQ ID NO: 6) concentrationstested were 41 nM; 123 nM; 370 nM; 1.1 uM; 3.3 uM; 10 uM. As shown inFIG. 18A, there is a moderate dose response for the high concentrations.

FIG. 18B presents a normalized cell index and a dose response curve forLGR7 transfected CHO-K1 cells challenged by increasing concentrations ofP59 (SEQ ID NO:6) at time point 26.5 hours (vertical right line in FIG.18A). A dose response is presented as a normalized cell index per Log ofconcentration (M). As can be seen from FIG. 18B a mild dose dependentactivation of LGR7 by P59C13V (SEQ ID NO:6) was found. Only the highestconcentration (10 uM) showed a significant higher cell index rate ascompared to the other concentrations. Since the same peptide batch wasused for this experiment and the experiment presented in FIG. 14supports the assumption that this particular batch of P59C13V peptidewas of bad quality. The affinity found for P59C13V (SEQ ID NO:6) to LGR7was significantly lower than that of H2 Relaxin or P74C13V (SEQ IDNO:10). Therefore, even though P59C13V (SEQ ID NO:6) peptide is capableof activating the receptor at high concentration, no real dose responsewas demonstrated.

FIG. 19A presents a normalized cell index and a dose response curve forLGR7 transfected CHO-K1 cells challenged by increasing concentrations ofpeptide P59S-Amide (P59S) (SEQ ID NO: 1) (the dashed Vertical linesindicates the 26^(th) hour from the beginning of the experiment). TheP59S (SEQ ID NO: 1) concentrations tested were 41 nM; 123 nM; 370 nM;1.1 uM; 3.3 uM; 10 uM. As shown in FIG. 19A, there is a moderate doseresponse for the high concentrations.

FIG. 19B presents a normalized cell index and a dose response curve forLGR7 transfected CHO-K1 cells challenged by increasing concentrations ofP59S-Amide (P59S) (SEQ ID NO: 1) at time point 26.5 hours (verticalright line in FIG. 18A). A dose response is presented as a normalizedcell index per Log of concentration (M). As can be seen from FIG. 19B, amild dose dependent activation of LGR7 by P59C13V (SEQ ID NO:6) wasfound. Only the highest concentration (10 uM) showed a significanthigher cell index rate as compared to the other concentrations.Therefore, even though this peptide is capable of activating thereceptor at high concentration, no dose response was demonstrated.

Example 8 The P59 Sequence in the C1QTNF8 Protein is Highly ConservedThroughout Other Species and Orthologs, as Well as at Least One HumanParalog (C1QTNF1)

FIG. 20 shows a multiple alignment comparison of the sequence of P59-G(SEQ ID No. 12), representing a fragment of the native human precursor(C1QTNF8—SEQ. ID. No. 19), and homologous sequences derived from variousorganisms, including Chimpanzee (SEQ ID No. 20), Orangutan (SEQ ID No.21), Rhesus (SEQ ID No. 22), Cow (SEQ ID No. 23), Chicken (SEQ ID No.24) and Rat (SEQ ID No. 26). The multiple sequence alignment comparisonincludes the corresponding peptide sequence derived from the humanparalogue C1QTNF1 (SEQ ID No. 25). As can be seen both the N-terminalend (4 Amino acids) and C-terminal end (7 Amino acids) are identical toall species. The cleavage sites at both ends are also highly conserved(with occasional replacement of K for an R). The middle Cysteine residue(C13) that was replaced with Valine for dimerization purposes is highlyconserved as well.

The descriptions given are intended to exemplify, but not limit, thescope of the invention. Additional embodiments are within the claims.

1-21. (canceled)
 22. A purified peptide less than 35 amino acids inlength, said peptide comprising an amino acid sequence of Formula I:X¹-X²-X³-X⁴-X⁵-X⁶-X⁷-X⁸-X⁹-X¹⁰-X¹¹-X¹²-X¹³-X¹⁴-X¹⁵-X¹⁶-X¹⁷-X¹⁸-X¹⁹-X²⁰-X²¹-X²²-X²³-X²⁴-X²⁵-X²⁶-X²⁷-X²⁸-X²⁹-X³⁰-X³¹-X³²-X³³wherein X¹ is absent or G or a small naturally or non-naturallyoccurring amino acid; X² is absent or Q or a polar naturally ornon-naturally occurring amino acid; X³ is absent or K or a basicnaturally or non-naturally occurring amino acid; X⁴ is absent or G or asmall naturally or non-naturally occurring amino acid; X⁵ is absent or Qor S a polar naturally or non-naturally occurring amino acid; X⁶ isabsent or V or A or P or M or a hydrophobic naturally or non-naturallyoccurring amino acid; X⁷ is absent or G or a small naturally ornon-naturally occurring amino acid; X⁸ is absent or P or L or A ornaturally or non-naturally occurring amino acid; X⁹ is absent or P or Qor a naturally or non-naturally occurring amino acid; X¹⁰ is absent or Gor a small naturally or non-naturally occurring amino acid; X¹¹ isabsent or A or H or E or D or a hydrophobic or a small or an acidicnaturally or non-naturally occurring amino acid; X¹² is absent or A or Por Q or S or R or H or a hydrophobic or a small naturally ornon-naturally occurring amino acid; X¹³ is absent or C or V or A or anyamino acid other than C; X¹⁴ is absent or R or K or Q or P or a basic ora polar naturally or non-naturally occurring amino acid; X¹⁵ is absentor R or Q or S or a basic or a polar naturally or non-naturallyoccurring amino acid; X¹⁶ is absent or A or L or H or Q or a hydrophobicor a small naturally or non-naturally occurring amino acid; X¹⁷ isabsent or Y or a hydrophobic or an aromatic naturally or non-naturallyoccurring amino acid; X¹⁸ is absent or A or a hydrophobic or smallnaturally or non-naturally occurring amino acid; X¹⁹ is absent or A or ahydrophobic small naturally or non-naturally occurring amino acid; X²⁰is absent or F or a hydrophobic or an aromatic naturally ornon-naturally occurring amino acid; X²¹ is absent or S or T or a polarnaturally or non-naturally occurring amino acid; X²² is absent or V or ahydrophobic naturally or non-naturally occurring amino acid; X²³ isabsent or G or hydrophobic or small naturally or non-naturally occurringamino acid or replaced by an Amide; X²⁴ is absent or R or a basicnaturally or non-naturally occurring amino acid; X²⁵ is absent or R or abasic naturally or non-naturally occurring amino acid; X²⁶ is A or ahydrophobic or small naturally or non-naturally occurring amino acid;X²⁷ is Y or a hydrophobic or an aromatic naturally or non-naturallyoccurring amino acid; X²⁸ is A or a hydrophobic or small naturally ornon-naturally occurring amino acid; X²⁹ is A or a hydrophobic or smallnaturally or non-naturally occurring amino acid; X³⁰ is F or ahydrophobic naturally or non-naturally occurring amino acid; X³¹ is S orT or a polar naturally or non-naturally occurring amino acid; X³² is Vor a hydrophobic naturally or non-naturally occurring amino acid; X³³ isabsent or G or hydrophobic or small naturally or non-naturally occurringamino acid or replaced by an amide; or a pharmaceutically acceptablesalt thereof.
 23. The peptide of claim 22, wherein said peptide bindsto/and or activates a GPCR in the LGR family of receptors or therelaxin-related family of receptors.
 24. The peptide of claim 22,wherein said peptide is isolated from a protein recombinantly producedin a prokaryotic or eukaryotic cell.
 25. The peptide of claim 22,wherein said peptide is chemically synthesized in vitro.
 26. The peptideof claim 22, wherein said peptide includes a C-terminal amidated aminoacid.
 27. The peptide of claim 22, wherein peptide is less than 30 aminoacids.
 28. The peptide of claim 22, wherein said peptide is less than 20amino acids.
 29. The peptide of claim 22, wherein said peptide isselected from the group consisting of: (SEQ ID NO: 1) AYAAFSV-Amide (SEQID NO: 2) AYAAFSV; (SEQ ID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; (SEQ IDNO: 4) GQKGQVGPPGAACRRAYAAFSV; (SEQ ID NO: 5)GQKGQVGPPGAAVRRAYAAFSV-Amide; (SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV;(SEQ ID NO: 7) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 8)GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 9)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 10)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 11) AYAAFSVG; (SEQ ID NO:12) GQKGQVGPPGAACRRAYAAFSVG; (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG;(SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; (SEQID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; (SEQ ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; (SEQID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; and (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG.


30. The peptide of claim 22, wherein said peptide is conjugated or fusedto a second peptide or polypeptide.
 31. A pharmaceutical compositioncomprising the peptide of claim 22 and a pharmaceutically acceptablecarrier.
 32. A method of treating a disorder or a condition selectedfrom the group consisting of hypertension associated disorder,cardiovascular disorder, fibrotic condition, endothelial dysfunctiondisease, respiratory disease, skin injury, condition associated withpregnancy, cancer, bone disease, ischemia-reperfusion injury,inflammatory disorder, autoimmune disorder, inflammatory conditionassociated with infection, kidney disease, and angiogenesis relatedcondition, the method comprising administering to said subject atherapeutically effective amount of a peptide of claim
 22. 33. Themethod of claim 32, wherein said peptide is selected from a groupconsisting of SEQ ID NOs: 1-15, 20-26.
 34. The method of claim 32,wherein the hypertension associated disorder is selected from a groupconsisting of hypertensive heart disease; antihypertension (bloodpressure reduction); systemic and pulmonary high blood pressure;cerebrovascular disease and stroke; heart failure and stroke; leftventricular hypertrophy (LVH); congestive heart failure (CHF);hypertension, high blood pressure; vasodilation; renal hypertension;diuresis; nephritis; natriuresis; scleroderma renal crisis; anginapectoris (stable and unstable); myocardial infarction; heart attack;coronary artery disease; coronary heart disease; cardiac arrhythmias;atrial fibrillation; portal hypertension; raised intraocular pressure;vascular restenosis; chronic hypertension; valvular disease; myocardialischemia; acute pulmonary edema; acute coronary syndrome; hypertensiveretinopathy; hypertensive pregnancy sickness; preeclampsia; Raynaud'sphenomenon; erectile dysfunction and glaucoma.
 35. The method of claim32, wherein the cancer is selected from a group consisting of coloncancer, lung cancer, breast cancer, prostate cancer, brain cancer,pancreatic cancer, ovarian cancer, kidney cancer, testicular cancer,bone cancer, osteosarcoma, liver cancer, melanoma, glioma, sarcoma,leukemia, or lymphoma, and wherein the cancer is invasive or metastatic.36. The method of claim 32, wherein the bone disease is selected from agroup consisting of osteoporosis; osteoarthritis; osteopetrosis; boneinconsistency; osteosarcoma; and cancer metastasis to the bone.
 37. Themethod of claim 32, wherein the ischemia-reperfusion injury isassociated with ischemic and post-ischemic events in organs and tissuesand is selected from a group consisting of thrombotic stroke; myocardialinfarction; angina pectoris; embolic vascular occlusions; peripheralvascular insufficiency; splanchnic artery occlusion; arterial occlusionby thrombi or embolisms, arterial occlusion by non-occlusive processessuch as following low mesenteric flow or sepsis; mesenteric arterialocclusion; mesenteric vein occlusion; ischemia-reperfusion injury to themesenteric microcirculation; ischemic acute renal failure;ischemia-reperfusion injury to the cerebral tissue; intestinalintussusception; hemodynamic shock; tissue dysfunction; organ failure;restenosis; atherosclerosis; thrombosis; platelet aggregation,ischemia-reperfusion injury following cardiac surgery; organ surgery;organ transplantation; angiography; cardiopulmonary and cerebralresuscitation.
 38. The method of claim 32, wherein the inflammatorycondition is associated with an infection, and is selected from thegroup consisting of a viral infection caused by human immunodeficiencyvirus I (HIV-1) or HIV-2, acquired immune deficiency (AIDS), West Nileencephalitis virus, coronavirus, rhinovirus, influenza virus, denguevirus, HCV, HBV, HAV, hemorrhagic fever; an otological infection; severeacute respiratory syndrome (SARS), sepsis and sinusitis.
 39. The methodof claim 32, wherein the inflammatory disorder is selected from thegroup consisting of gastritis, gout, gouty arthritis, arthritis,rheumatoid arthritis, inflammatory bowel disease, Crohn's disease,ulcerative colitis, ulcers, chronic bronchitis, asthma, allergy, acutelung injury, pulmonary inflammation, airway hyper-responsiveness,vasculitis, septic shock and inflammatory skin disorders, including butnot limited to psoriasis, atopic dermatitis, eczema.
 40. An antibodythat selectively binds to an epitope in the peptide of claim
 22. 41. Theantibody of claim 39, wherein said peptide is selected from the groupconsisting of (SEQ ID NO: 1) AYAAFSV-Amide (SEQ ID NO: 2) AYAAFSV; (SEQID NO: 3) GQKGQVGPPGAACRRAYAAFSV-Amide; (SEQ ID NO: 4)GQKGQVGPPGAACRRAYAAFSV; (SEQ ID NO: 5) GQKGQVGPPGAAVRRAYAAFSV-Amide;(SEQ ID NO: 6) GQKGQVGPPGAAVRRAYAAFSV; (SEQ ID NO: 7)GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 8)GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 9)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV-Amide; (SEQ ID NO: 10)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSV; (SEQ ID NO: 11) AYAAFSVG; (SEQ ID NO:12) GQKGQVGPPGAACRRAYAAFSVG; (SEQ ID NO: 13) GQKGQVGPPGAAVRRAYAAFSVG;(SEQ ID NO: 14) GQKGQVGPPGAACRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 15)GQKGQVGPPGAAVRRAYAAFSVGRRAYAAFSVG; (SEQ ID NO: 20)GQKGQVGPPGAACQRAYAAFSVG; (SEQ ID NO: 21) GQKGQVGPPGAPCQRAYAAFSVG; (SEQID NO: 22) GQKGQVGPPGAPCQRAYAAFSVG; (SEQ ID NO: 23)GQKGQAGLPGAQCPRAYAAFSVG; (SEQ ID NO: 24) GQKGQPGPQGHSCKQLYAAFSVG; (SEQID NO: 25) GQKGSMGAPGERCKSHYAAFSVG; and (SEQ ID NO: 26)GQKGSMGAPGDHCKSQYAAFSVG.